CN107586743B - Bacillus megaterium capable of efficiently dissolving phosphorus at root zone of forest trees and application thereof - Google Patents
Bacillus megaterium capable of efficiently dissolving phosphorus at root zone of forest trees and application thereof Download PDFInfo
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Abstract
The invention discloses a forest rhizosphere high-efficiency phosphorus-solubilizing Bacillus megaterium with a preservation number of CCTCC NO.M 2017454, which is named as Bacillus megaterium (Bacillus megaterium) L205 by classification. Tests show that the phosphorus-dissolving amount of the bacillus megaterium reaches 203.04mg/L, and the liquid bacterial liquid of the bacillus megaterium can effectively dissolve insoluble inorganic phosphorus, increase the effective phosphorus content in the red-yellow soil and promote the growth of forest seedlings. The invention enriches the population library of the bacillus megaterium in the strain preservation center and provides convenience for better developing and utilizing microbial population resources of the bacillus megaterium. Meanwhile, the bacillus megaterium strain L205 can secrete organic acid, dissolve inorganic phosphorus and improve the absorption and utilization of phosphorus by plants, is particularly suitable for the production of forest biological bacterial fertilizers and forest growth promoting microbial inoculum in red and yellow soil areas in south China, and can be further developed and developed into microbial compound fertilizers and microbial inoculum.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a forest rhizosphere high-efficiency phosphorus-dissolving bacillus megaterium and application thereof.
Background
The rhizosphere is a place where the soil, the root system and the microorganism are tightly combined and mutually influenced, and is also an important component of a soil microecological system. Sperber et al found that most of phosphate solubilizing microorganisms appear in plant rhizosphere soil, and rhizosphere phosphate solubilizing bacteria and root systems have mutual promotion effects. Most of soil in China is in a phosphorus deficiency state, more than 95% of phosphorus in soil in south China is indissolvable phosphorus and cannot be directly absorbed by plants, and phosphorus becomes a main factor influencing plant growth. Meanwhile, most of the southern fast-growing and high-yield forests are artificial pure forests, the biological diversity is simple, the ecological system is weak, and the problems of reduced ecological environment quality, water and soil pollution, soil hardening and the like caused by the application of a large amount of unreasonable chemical fertilizers are increasingly serious.
The forest land has the advantages that the number of dead branches and fallen leaves is large, organic matters are rich, soil microorganisms are more abundant relative to agricultural soil, and according to the research of the past years, the proportion of the number of bacteria in phosphorus-dissolving microorganisms in dry land soil in China is the largest. The currently reported types of phosphate solubilizing bacteria mainly include: bacillus (Bacillus), Pseudomonas (Pseudomonas), Salmonella (Salmonella), Alcaligenes (Alcaligenes) and the like, wherein Bacillus megaterium (Bacillus megaterium) is the strain which is found to have the earliest and the best phosphorus solubilizing effect and has the widest agricultural application area.
Different soil environment climatic characteristics and different plant rhizosphere conditions are different, so that the special soil environment can cause the defects of poor colonization ability of non-indigenous strains, high strain elimination rate and the like. The soil in Guangxi province is mainly red yellow soil, and the red yellow soil is also a typical soil type in the south of China. Few reports about rhizosphere phosphate solubilizing bacteria of forest trees in red and yellow soil areas in China exist. Due to the special soil forming process, clay, iron oxide and aluminum oxide in the soil are high, and available phosphorus is deficient, so that the increase of the forest storage amount in the area is restricted. The high-efficiency phosphorus-dissolving bacterium Y5 is obtained by separating Dashenyan and the like from Jiangxi Yingtan rice soil, and the phosphorus-dissolving amount of the high-efficiency phosphorus-dissolving bacterium Y5 to calcium phosphate is 159.1 mg/L. At present, the research on rhizosphere phosphorus-dissolving bacteria of red-yellow soil trees in forest lands is less, and the screened phosphorus-dissolving bacteria are all derived from cultivated land crop soil, so that the application effect on the production of the trees is limited.
Disclosure of Invention
The invention aims to solve the technical problem of providing a forest rhizosphere high-efficiency phosphorus-dissolving bacillus megaterium which has good phosphorus-dissolving effect, strong colonization ability, wide application range and low production cost and an application thereof, wherein the strain is particularly suitable for the production of forest biological bacterial fertilizers and forest growth-promoting bactericides in red and yellow soil regions in south China.
In order to solve the technical problems, the invention adopts the following technical scheme:
a strain of high-efficiency rhizosphere phosphorus-solubilizing Bacillus megaterium for forest has a preservation number of CCTCC N0.M 2017454 and is classified and named as Bacillus megaterium L205.
The application of the high-efficiency phosphorus-dissolving bacillus megaterium at the rhizosphere of forest in dissolving insoluble inorganic phosphorus.
The insoluble inorganic phosphorus is from red yellow soil.
The insoluble inorganic phosphorus is calcium phosphate.
In the application, the propagation conditions of the bacillus megaterium are that the temperature is 15-35 ℃, the pH value is 6.0-9.0, the dissolved oxygen is 0.2-2.0 mg/L, and the mass ratio of carbon to nitrogen is 2: 1-5: 1.
The application of the bacillus megaterium for efficiently dissolving phosphorus in the rhizosphere of the trees in promoting the growth of the nursery stocks of the trees.
The forest nursery stock grows in the red and yellow soil.
In the application, the propagation conditions of the bacillus megaterium are that the temperature is 15-35 ℃, the pH value is 6.0-9.0, the dissolved oxygen is 0.2-2.0 mg/L, and the mass ratio of carbon to nitrogen is 2: 1-5: 1.
The high-efficiency phosphorus-dissolving bacillus megaterium at the rhizosphere of the forest is used for producing forest biological bacterial fertilizer or forest growth-promoting microbial inoculum.
The total number of the bacillus megaterium L205 or the mixed bacteria is not less than 0.2 x 108cfu/mL(g)。
The inventor obtains an effective phosphorus-solubilizing strain by separating and breeding from forest rhizosphere soil in red-yellow soil region in Guangxi, and the strain is identified as bacillus megaterium by performing Blast comparison on 16SrDNA and a sequence in GenBank. The forest rhizosphere efficient phosphate-solubilizing Bacillus megaterium has a preservation number of CCTCC N0.M 2017454 and is named as Bacillus megaterium (Bacillus megaterium) L205 in a classified manner. In the research of the application of the strain, the inventor obtains the strain propagation process technology by pure culture propagation and liquid fermentation process optimization. Tests show that the phosphorus-dissolving amount of the bacillus megaterium reaches 203.04mg/L, and the liquid bacterial liquid of the bacillus megaterium can effectively dissolve insoluble inorganic phosphorus, increase the effective phosphorus content in the red-yellow soil and promote the growth of forest seedlings. The phosphorus-solubilizing strain enriches the population library of the bacillus megaterium in the strain preservation center, and provides convenience for better developing and utilizing microbial population resources of the bacillus megaterium. Meanwhile, the bacillus megaterium strain L205 can secrete organic acid, dissolve inorganic phosphorus and improve the absorption and utilization of phosphorus by plants, is particularly suitable for the production of forest biological bacterial fertilizers and forest growth promoting microbial inoculum in red and yellow soil areas in south China, and can be further developed and developed into microbial compound fertilizers and microbial inoculum. In a word, the high-efficiency phosphorus-dissolving bacillus megaterium for forest rhizosphere has the advantages of good phosphorus-dissolving effect, strong colonization capability, wide application range, low production cost and larger market application potential.
Drawings
FIG. 1 is a phylogenetic tree of Bacillus megaterium L205 of the present invention.
FIG. 2 is a graph showing the results of phosphorus solubilization of Bacillus megaterium L205 of the present invention, in which: front view of the plate A and back view of the plate B.
Description of preservation information
Bacillus megaterium (Bacillus megaterium) L205 with a preservation number of CCTCC No. m 2017454, preservation date: 28/8/2017, the preservation address is as follows: eight-way Lojia mountain in Wuchang district, Wuhan city, Hubei province, post code 430072, preservation unit: china center for type culture Collection.
Detailed Description
The present invention will be described in further detail with reference to the following examples and the accompanying drawings.
Bacterial strain source, breeding and gene sequencing of bacillus megatherium
The strain source is as follows: the Bacillus megaterium L205 is obtained by separating and breeding from forest rhizosphere soil in red and yellow soil region in Guangxi, and the preservation information is the same as above.
The morphological characteristics of the bacillus megaterium L205 are as follows: the colony is white, the thallus is rod-shaped, and has spores.
The physiological and biochemical characteristics of the bacillus megaterium L205 are as follows: gram staining is positive, catalase positive, oxidase positive.
The bacillus megaterium L205 is obtained by the following specific isolation steps:
collecting soil A: collecting the rhizosphere soil of trees with good growth vigor in areas of Guangxi red and yellow soil, such as Sterculia, Ningming, Luzhai, Tianlin and the like, taking 0-20 cm rhizosphere non-rhizosphere soil and root system samples, filling the rhizosphere non-rhizosphere soil and the root system samples into a fresh-keeping bag which is not unsealed, sticking a label, sealing, placing the fresh-keeping bag in an ice bag, and cutting the fresh-keeping bag back to a laboratory for phosphorus-solubilizing strain separation.
The results of the measurements of the various soil samples are shown in Table 1. As can be seen from Table 1: the organic matter content of the soil samples collected from Guangxi Sterculia, Ningming, Anzhai and Tianlin is more than 36.75mg/kg, the organic matter content of the four soil samples is 36.75-56.51mg/kg, and the soil sample with the lowest organic matter content is collected from Anzhai. The total phosphorus content of the soil collected from Sterculia is the highest and is 1.48g/kg, and the quick-acting content of the soil of a sample collected from a field is the highest and is 16.53 mg/kg. The pH value of each soil sample is between 4.15 and 5.02 and is slightly acidic.
TABLE 1 soil sample analysis results from four different areas in Guangxi
B, strain separation: 5g of eucalyptus rhizosphere soil samples in different areas are weighed, placed in a sterilized 150mL triangular flask filled with 45mL of 0.85% NaCl solution, and shaken for 30min to prepare suspensions. Sucking 1mL of suspension from a 1mL microsyringe, injecting the suspension into a test tube containing 9mL of 0.85% sterile NaCl solution, mixing uniformly, sucking 1mL from the test tube, injecting the suspension into another test tube containing 9mL of 0.85% sterile NaCl solution, and so on to obtain the final product with the content (g) and NaCl solution (mL) ratio of 10-3、10-4、10-53 kinds of dilution ladderSoil suspension of the degree. Pipette 50 μ L of coating, repeat 3 per gradient, and mark well. And (3) keeping the temperature at 28 ℃, culturing for 2-5D, screening strains with phosphorus-dissolving rings on a culture medium of phosphorus-dissolving bacteria, determining the phosphorus-dissolving capacity according to the HE value which is the ratio of the diameter (D) of the phosphorus-dissolving rings to the diameter (D) of bacterial colonies, respectively inoculating the strains with stronger phosphorus-dissolving capacity to an LB plate for purification, then storing the strains on an LB inclined plane culture medium, and keeping the strains in a refrigerator at 4 ℃ for later use.
C, phosphorus dissolving capacity determination: 100mL of liquid PKO culture medium is filled into a 250mL triangular flask, sterilized at 121 ℃ for 20min, and after cooling, 1mL of each bacterial suspension (10) of the bacterial strain to be detected is added8cfu/mL) into triangular flasks. Each strain was set to 3 replicates and the control was replaced with 1ml sterile water. The flask was shake-cultured at 28 ℃ and 160r/min for 3 days, and the pH of the culture was measured with a pH meter. Centrifuging at 4 deg.C 10000 r/min for 10min, collecting supernatant 5mL, and determining effective phosphorus content at wavelength of 700nm by molybdenum-antimony-scandium colorimetry.
D, liquid culture: selecting a ring of bacillus megaterium from a single colony test tube slant, inoculating the bacillus megaterium in a liquid culture medium, culturing the bacillus megaterium for 24 hours at 32 ℃ on a shaker with the rotation speed of 150 plus 220rpm, wherein the viable count reaches 6 multiplied by 108About cfu/mL.
E, strain preservation: and (3) adding glycerol subjected to high-temperature sterilization into the bacillus megaterium liquid obtained by fermenting the liquid to ensure that the final concentration of the glycerol in the seed liquid is 20% (V/V), preparing a bacillus megaterium glycerol freezing tube, and storing the bacillus megaterium glycerol freezing tube in a refrigerator at minus 80 ℃ or in a liquid nitrogen tank at minus 196 ℃ for ultralow-temperature storage.
F gene sequence determination: the bacillus megaterium test tube inclined plane is sent to Shanghai Meiji biotechnology company for 16sRNA gene sequencing, and the 16s rRNA gene sequence is shown as a base sequence of a sequence table SEQ.ID.No. 1. Its phylogenetic tree is shown in FIG. 1.
Phosphorus dissolution test
PKO (Pikovskaya's Medium) Medium: 10g of glucose, 3g of calcium phosphate, 0.5g of ammonium sulfate, 0.1g of sodium chloride, 0.1g of magnesium sulfate heptahydrate, 0.2g of potassium chloride, 0.004g of manganese sulfate, 0.002g of ferrous sulfate, 0.5g of yeast extract and 1000ml of water, and the pH value is 7.2.
The bacillus megaterium CCTCC M2017454 is inoculated on a PK0 culture medium, cultured for 5D at the temperature of 30 ℃, and the ratio of the diameter (D) of a phosphorus-dissolving ring, namely a transparent ring to the size (D) of a colony is determined to be 2.67. The bacillus megaterium is proved to have good phosphorus dissolving effect. Measurement of phosphorus dissolution
PKO (Pikovskaya's Medium) Medium: 10g of glucose, 3g of calcium phosphate, 0.5g of ammonium sulfate, 0.1g of sodium chloride, 0.1g of magnesium sulfate heptahydrate, 0.2g of potassium chloride, 0.004g of manganese sulfate, 0.002g of ferrous sulfate, 0.5g of yeast extract and 1000ml of water, and the pH value is 7.2.
100mL of liquid PK0 culture medium is filled into a 250mL triangular flask, sterilized at 121 ℃ for 20min, and after cooling, 1mL of Bacillus megaterium CCTCC M2017454 (10)8cfu/mL) into triangular flasks. Each strain was set to 3 replicates and the control was replaced with 1ml sterile water. The flask was shake-cultured at 28 ℃ and 160r/min for 3 days, and the pH of the culture was measured with a pH meter. Centrifuging at 4 deg.C 10000 r/min for 10min, collecting supernatant 5mL, and determining effective phosphorus content at wavelength of 700nm by molybdenum-antimony-scandium colorimetry.
The phosphorus dissolving capacity of the L205 strain under the liquid shake flask culture condition shows that the effective phosphorus increment of the strain for dissolving inorganic phosphorus bacteria is 203.04 mg/L.
Preparation of forest phosphate-solubilizing microbial liquid
(1) Seed preparation
Seed culture medium: 10g of tryptone, 5g of yeast extract powder, 10g of NaCl, 7.0 of pH value and 1000mL of distilled water. Inoculating bacillus megaterium CCTCC M2017454 into a 250mL triangular flask containing 100mL of the seed culture medium for 1 circle, and culturing at 30 ℃ for 24 hours at the rotating speed of 180rpm of a rotary shaking table to obtain seeds suitable for inoculation.
(2) Liquid fermentation culture
Fermentation medium: 10g of tryptone, 5g of yeast extract powder, 10g of NaCl, 7.0 of pH value and 1000mL of distilled water. Inoculating the bacillus megaterium seeds obtained in the step (1) into a liquid fermentation culture medium according to 5% of the volume of the fermentation culture medium, and culturing for 48h at 30 ℃ at the rotating speed of 180rpm of a rotary shaking table.
(3) After the fermentation is finished, the pH value of the fermentation liquor is adjusted to be between 5.0 and 6.0, and then the liquid microbial inoculum can be obtained.
Preparation of microbial agent for phosphorus dissolving of trees
(1) Seed preparation
Seed culture medium: 10g of tryptone, 5g of yeast extract powder, 10g of NaCl, 7.0 of pH value and 1000mL of distilled water. Inoculating bacillus megaterium CCTCC M2017454 into a 250mL triangular flask containing 100mL of the seed culture medium for 1 circle, and culturing at 30 ℃ for 24 hours at the rotating speed of 180rpm of a rotary shaking table to obtain seeds suitable for inoculation.
(2) Liquid fermentation culture
Fermentation medium: 10g of tryptone, 5g of yeast extract powder, 10g of NaCl, 7.0 of pH value and 1000mL of distilled water. Inoculating the bacillus megaterium seeds obtained in the step (1) into a liquid fermentation culture medium according to 5% of the volume of the fermentation culture medium, and culturing for 48h at 30 ℃ at the rotating speed of 180rpm of a rotary shaking table.
(3) And after the fermentation is finished, placing the liquid fermentation culture medium in a high-speed centrifuge for centrifugation for 5 minutes, removing supernatant, adding 1% of Tween 80, and freeze-drying to obtain the forest phosphate-solubilizing microbial agent.
Application of forest phosphorus-dissolving microbial liquid in cultivation of cup seedlings of forest trees
And (4) diluting the bacterial liquid obtained in the step four by 10 times by using sterile water by adopting a root irrigation inoculation method. And (3) taking pine and cedar seedlings with the same growth vigor, irrigating 10mL of diluted bacterial liquid, irrigating 50mL of sterile water every 2 days, and applying phosphorus-deficient Hoagland nutrient solution every 10 days. 25 plants are treated at each concentration, clear water is used as a control, and the test temperature is 15-30 ℃. And after 30 days of inoculation, calculating the survival number of each treated seedling, the average seedling height of the plant and the ground diameter.
The measurement result shows that: the cup seedling of pine tree added with the phosphate-solubilizing microbial agent has a gain of 12.3 percent and a gain of 10.6 percent of land diameter compared with the control group seedling, the cup seedling of fir tree has a gain of 9.5 percent and a gain of 12.8 percent of land diameter compared with the control group seedling. The microbial preparation of the invention has better growth promoting effect on forest trees.
Sequence listing
<110> Guangxi Zhuang nationality autonomous region forestry science research institute
<120> forest rhizosphere high-efficiency phosphorus-dissolving bacillus megaterium and application thereof
<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 strain of high-efficiency rhizosphere phosphorus-solubilizing Bacillus megaterium for forest is characterized in that the preservation number is CCTCC NO.M 2017454, and the strain is classified and named as Bacillus megaterium (Bacillus megaterium) L205.
2. The application of the rhizosphere bacillus megaterium for efficiently dissolving phosphorus in forest as claimed in claim 1 in dissolving insoluble inorganic phosphorus.
3. Use according to claim 2, characterized in that: the slightly soluble inorganic phosphorus is from red yellow soil.
4. Use according to claim 2, characterized in that: the insoluble inorganic phosphorus is calcium phosphate.
5. The application of the rhizosphere bacillus megaterium karphosphate solution for promoting the growth of nursery stocks of forest trees as claimed in claim 1.
6. Use according to claim 5, characterized in that: the forest nursery stock grows in red and yellow soil.
7. The application of the rhizosphere bacillus megaterium for efficient phosphorus solubilization of forest as defined in claim 1 in the production of forest biological bacterial fertilizer or forest growth promoting microbial inoculum.
8. Use according to claim 7, characterized in that: the total number of bacillus megaterium L205 in the forest biological bacterial fertilizer or the forest growth promoting microbial inoculum is not less than 0.2 x 108cfu/mL or 0.2 x 108cfu/g。
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| CN108676744B (en) * | 2018-05-16 | 2020-07-28 | 南京农业大学 | Bacillus megaterium strain ZT-P and application thereof |
| CN110684696B (en) * | 2019-11-11 | 2022-04-19 | 安顺学院 | Bacillus megaterium QZY-3 and application thereof |
| CN111676164B (en) * | 2020-06-19 | 2022-05-20 | 中国林业科学研究院林业研究所 | Phosphate solubilizing bacterium RP2, and fermentation product, microbial inoculum and application thereof |
| CN112852687A (en) * | 2021-04-15 | 2021-05-28 | 河南科技学院 | Peony rhizosphere bacillus megatherium FD3-13 for three-year-old oil and application |
| CN114574395A (en) * | 2022-03-17 | 2022-06-03 | 广西壮族自治区林业科学研究院 | Bacillus megaterium and biofertilizer capable of antagonizing colletotrichum gloeosporioides |
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| CN103320360A (en) * | 2013-06-25 | 2013-09-25 | 云南省烟草公司玉溪市公司 | Bacillus megatherium SJ-7 strain and application thereof |
| CN103992963A (en) * | 2014-03-19 | 2014-08-20 | 南京博农生物科技有限公司 | Bacillus megaterium and application thereof |
| CN104560787A (en) * | 2014-12-13 | 2015-04-29 | 郑州市污水净化有限公司 | Peanut rhizosphere growth-promoting bacteria HS11 and application thereof |
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| CN103320360A (en) * | 2013-06-25 | 2013-09-25 | 云南省烟草公司玉溪市公司 | Bacillus megatherium SJ-7 strain and application thereof |
| CN103992963A (en) * | 2014-03-19 | 2014-08-20 | 南京博农生物科技有限公司 | Bacillus megaterium and application thereof |
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