CN114437965B

CN114437965B - Peanut rhizosphere microbial strain and method for improving and repairing soil by using biological bacterial fertilizer thereof

Info

Publication number: CN114437965B
Application number: CN202111677070.7A
Authority: CN
Inventors: 孙伟明; 冯丽娜; 邢单润; 胡朋举; 焦镇; 李步阳; 宋亚辉
Original assignee: Hebei Normal University of Science and Technology
Current assignee: Jing'an Ecological Technology Group Co ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-12-20
Anticipated expiration: 2041-12-31
Also published as: CN114437965A

Abstract

The invention provides a method for improving and repairing soil by using a peanut rhizosphere microbial strain and a biological bacterial fertilizer thereof, and relates to the technical field of soil improvement. The invention comprises strains which have an inhibiting effect on pathogenic bacteria of peanut fruit rot, strains which have the capabilities of dissolving phosphorus, dissolving potassium, fixing nitrogen, adjusting pH and degrading peanut autotoxic substances and strains which secrete plant growth regulators. The strains are compounded and applied to peanut planting, and the peanut seedlings have remarkable growth-promoting, disease-preventing and yield-increasing effects. Meanwhile, the biological bacterial fertilizer prepared by the peanut rhizosphere microbial bacterial line is applied by matching the microbial bacterial line, organic matters and inorganic compound fertilizers, so that the peanut rot is prevented and treated, the yield is increased by the synergistic effect of growth-promoting bacteria and the growth-promoting effect of the biological organic fertilizer, and the yield is improved by preventing and treating the peanut rot through the synergistic effect of biocontrol strains and growth-promoting strains.

Description

Peanut rhizosphere microbial strain and method for improving and repairing soil by using biological bacterial fertilizer thereof

Technical Field

The invention belongs to the technical field of soil improvement, and particularly relates to a peanut rhizosphere microbial strain and a method for improving and repairing soil by using prepared biological bacterial fertilizer.

Background

Peanuts are one of main oil crops and economic crops in China, peanut rot is a commonly occurring soil-borne disease in the world, and is reported in North America, australia, asia, africa and other places. In recent years, peanut rot occurs in peanut main producing areas such as Hebei, henan, shandong, liaoning and the like, and the peanut rot has a tendency of increasing year by year and becomes a main disease of the peanut main producing areas in China. Peanut rot can cause about 30% of peanut pod rot, and seriously threatens the healthy development of the flower production industry.

Continuous cropping (continuous cropping obstacle) can aggravate the occurrence of peanut rot, resulting in over 50% yield reduction and even top harvest. The main reasons for aggravating peanut rot by continuous cropping (continuous cropping obstacle) are as follows: firstly, excessive application of fertilizers with high nitrogen, phosphorus and potassium, application of organic matters lack, and improper supplement of mineral elements such as calcium, iron, magnesium and the like cause unbalance of soil nutrient elements. In general, sufficient nitrogen, phosphorus and potassium are accumulated in the peanut continuous cropping land, and sufficient organic matters and mineral elements such as calcium, iron, magnesium and the like which can be absorbed and utilized are lacked. Secondly, benzoic acid, p-hydroxybenzoic acid, vanillic acid, coumaric acid and other phenolic acids secreted by the peanut root system are induced by the accumulation of a large amount of toxic substances, so that the normal growth of the peanuts is inhibited. And thirdly, beneficial soil bacteria such as bacillus and streptomyces are continuously reduced, pathogenic microorganisms such as fusarium are continuously accumulated, so that the species and the quantity of the probiotics and the harmful bacteria of the soil microorganisms are unbalanced, and the restriction and inhibition effects of the soil on the pathogenic microorganisms are reduced. Fourthly, soil acidification is carried out, the utilization of key nutrient elements by plants or certain microorganisms is influenced, meanwhile, the soil acidification can change the composition of soil microbial communities, destroy the biological barriers of plants and is beneficial to the pathogenicity of pathogenic bacteria. In a word, the growth of pods and roots of the peanuts in the continuous cropping land is hindered due to the four factors, soil-borne diseases and pests such as peanut rot and root rot are aggravated year by year, and a special soil remediation modifier for the continuous cropping land of the peanuts is urgently needed to cultivate healthy soil and reduce the occurrence of the soil-borne diseases and pests, so that the purpose of high quality and high yield is achieved.

At present, the main cultivated species of peanuts in China are generally susceptible to peanut rot, and particularly, no high-oleic-acid species has a high resistance which can be popularized. Although the seed quality resources of peanuts at home and abroad are evaluated for resisting fruit rot by adopting a natural disease nursery identification method in the areas of He Meijing and the like, 2 parts of high-resistance seed quality resources are obtained, and a longer path is provided for the production of the disease-resistant variety cultivated at a distance. The chemical agent has the defects of short drug effect period, environmental friendliness and the like in the aspect of preventing and treating soil-borne diseases. The biological control can make up the defects and has good application prospect in the control of soil-borne diseases. At present, some excellent strains are reported in the biological control of peanut rot, but the disease prevention effect of biocontrol microorganisms in the field control practice is unstable and nonuniform, so that no medicine is available for the disease in the actual production. This is because the biocontrol strain only considers the inhibition effect on pathogenic bacteria, but does not consider the soil microenvironment for survival and the interaction with other microorganisms or plants, so that the biocontrol strain cannot be colonized in the field condition, and even after partial colonization, the soil environment on which the pathogenic bacteria live is not changed, and the synergistic effect of microorganisms, crops and soil is not promoted. Therefore, a microbial-organic-inorganic combined preparation is needed, which not only can change the soil microenvironment to enable beneficial microbes to effectively colonize, but also can interact with peanuts to promote healthy development.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for improving and repairing soil by using a peanut rhizosphere microbial strain and a biological bacterial fertilizer thereof, which can effectively reduce the incidence rate of peanut rot in continuous cropping plots, increase the yield of peanuts, and reduce pesticide residues caused by excessive application of fertilizers and pesticides, thereby realizing disease prevention, fruit preservation and yield increase.

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

the invention provides Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) GF-32a, wherein the strain GF-32a is preserved with the preservation number of CGMCC No.22902.

Preferably, the 16S rDNA sequence of the strain GF-32a is shown in SEQ ID NO. 1.

The invention also provides a microbial strain for improving the peanuts in the continuous cropping land, wherein the microbial agent comprises the strain GF-32a, bacillus megaterium GD-16 and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) GF-3.

Preferably, the viable count ratio of the strain GF-32a, the strain GD-16 and the strain GF-3 is 0.05-0.1 hundred million/g: 0.2-0.5 hundred million/g: 0.5-1.0 hundred million/g.

Preferably, the microbial fertilizer (biological bacterial fertilizer (powdery peanut special soil improvement and restoration agent)) also comprises the following raw materials in percentage by mass: 75.0 to 85.0 percent of solid fermentation material of livestock and poultry manure, 3.0 to 5.0 percent of sugarcane filter mud, 3.0 to 5.0 percent of humic acid, 8.0 to 10.0 percent of potassium sulfate, 3.0 to 5.0 percent of limestone powder and 1.0 to 3.0 percent of plant ash.

The invention also provides application of the strain GF-32a or the microbial fertilizer in improvement and remediation of peanut soil.

The invention also provides a method for improving and repairing the continuous cropping peanut soil, which comprises the following steps: before peanut sowing, the microbial agent is broadcast into a sowing field.

Preferably, the spreading amount of the microbial agent is 40 to 80kg/mu.

Preferably, the application further comprises the step of applying an inorganic compound fertilizer by 20 to 30kg/mu.

Preferably, the method further comprises the steps of turning the soil deeply for 25 to 30cm after the sowing, turning the microbial agent into the soil, and applying the inorganic compound fertilizer in a ditch during sowing.

Has the advantages that: the invention develops a biological bacterial fertilizer (powder type special soil improvement and restoration agent for peanuts), which comprises bacterial strains with an inhibiting effect on pathogenic bacteria of peanut rot, bacterial strains with the functions of dissolving phosphorus, dissolving potassium, fixing nitrogen, adjusting pH and degrading autotoxic substances of peanuts and bacterial strains secreting a plant growth regulator. The strains are compounded and applied to peanut planting, and have remarkable growth promoting, disease preventing and yield increasing effects compared with the strains which have the effect of preventing and treating the fruit rot alone. Meanwhile, the biological bacterial fertilizer (powder type special soil improvement and restoration agent for peanuts) is used for preventing and treating peanut rot by matching and applying a microbial strain, an organic matter and an inorganic compound fertilizer, so that the yield can be increased by the synergistic effect of growth-promoting bacteria and the growth-promoting effect of the biological organic fertilizer, and the yield can be increased by preventing and treating the peanut rot through the synergistic effect of biocontrol strains and growth-promoting strains. Compared with chemical pesticides (suspension seed coating agents, the seed dressing agent of the chemical pesticides is set as blank control in the invention) and microbial fertilizers (such as bacillus amyloliquefaciens), the microbial fertilizer has obvious, uniform and stable control effect and yield increase effect, and the synergistic effect is shown among the strains.

The concrete test shows that:

(1) In the pot experiment, the complex microbial inoculant prepared by using the GF-32a, the GD-16 and the GF-3 has the effect of remarkably promoting the growth of the main root compared with single microbial treatment.

(2) The cell test results show that: the biological bacterial manure (powder type peanut special soil improvement and restoration agent) prepared by the compound microbial agent is better than a conventional control (a biological organic fertilizer prepared by GF-32a, GD-16 or GF-3 independently) and a blank control (chemical agents) in the aspects of emergence rate and yield.

(3) The test result of field peanut rot prevention and control shows that: the seed dressing Jihua No. 16 (blank control) peanut rot disease caused by using a suspended seed coating agent (3% fludioxonil and 3% thifluzamide) is serious, the disease indexes in two years are respectively 36.49 +/-3.82 and 33.46 +/-4.15, and the field control effect on the peanut rot disease by using chemical agent treatment (blank control) is very limited.

The prevention and treatment efficiency of the biological bacterial manure (the powdery special soil improvement and restoration agent for peanuts) on peanut rot is as high as 90.50% +/-1.15% to 96.23% +/-1.78%, the yield increase rate is as high as 41.39% +/-3.11% to 42.34% +/-5.63%, the prevention and treatment effect and the yield increase effect are remarkably better than other treatment effects, and the effect is stable. In a word, the biological bacterial manure (the powder type special soil improvement and restoration agent for peanuts) has good prevention and control effects and yield increase effects on the peanut rot of the stubble lands.

The biological bacterial fertilizer (the powder type special soil improvement and restoration agent for peanuts) improves the microbial community structure in soil, reduces the abundance of pathogenic fungi, can reduce the infection of pathogenic bacteria on peanuts and is beneficial to reducing the morbidity. Meanwhile, the utility model is beneficial to fineness.

Biological preservation

Paenibacillus mucilaginosus (A), (B)Paenibacillus mucilaginosus) The strain is named as GF-32a, and has been preserved in China general microbiological culture Collection center at 7 month and 15 days 2021, with the preservation address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101, accession No.22902; the preservation number is CGMCC No.22902;

megaspore Bacillus (A), (B)Bacillus megaterium) The strain is named GD-16, has been preserved in China general microbiological culture Collection center in 30 months 12 and 2021, and the preservation address is as follows: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101; the preservation number is CGMCC No.24218;

bacillus amyloliquefaciens (A), (B), (C), (B) and (C)Bacillus amyloliquefaciens) The strain is named as GF-3 and published in microbiological newspaper in 2018, 2, 7, and the article is named as separation identification and disease prevention effect of peanut pulse-invading red shell fungus fruit rot biocontrol bacillus.

Drawings

FIG. 1 shows the colony morphology of the GF-32a strain;

FIG. 2 shows the cell morphology of the GF-32a strain;

FIG. 3 shows the results of preliminary verification of phosphorus-solubilizing potassium-fixing nitrogen by GF-32a strain;

FIG. 4 shows the results of preliminary verification of the degradation of peanut autotoxic substances by the strain GF-32 a;

FIG. 5 shows the structural changes of the phylum level fungal community in soil;

FIG. 6 shows structural changes in fungal community at the genus level in soil.

Detailed Description

One of the invention provides Paenibacillus mucilaginosus (GF-32 a), which is preserved in China General Microbiological Culture Center (CGMCC), the preservation date is 2021 year, 7 month and 15 days, and the preservation number is CGMCC No.22902.

The invention also provides a method for improving and repairing peanuts in a microbial system-organic-inorganic 'three-in-one' continuous cropping field, which comprises the step of forming a compound microbial system with complementary functions by the bacillus mucilaginosus GF-32a, the bacillus megaterium GD-16 and the bacillus amyloliquefaciens GF-3, and the compound microbial system can be colonized in rhizosphere, root surface and root to form a 'three-in-one' functional microbial protective bacterial system. The method also comprises a three-in-one combination of a microbial strain, solid fermentation organic matters and mineral elements required by peanuts, and is used for improving the physical and chemical properties of soil, adjusting nutrient substances required by crops, increasing beneficial microorganisms, controlling harmful microorganisms, adjusting microbial flora, degrading autotoxic substances of peanut root systems, improving the immunity of the peanuts, and achieving the effects of promoting growth and increasing yield, resisting continuous cropping and preventing and controlling peanut rot.

The paenibacillus mucilaginosus is applied to colonizing root system soil and has at least one of functions of degrading peanut autotoxic substances, dissolving phosphorus, dissolving potassium, adjusting pH and the like, so that the micro-ecological environment of rhizosphere soil is improved, and continuous cropping obstacles are reduced. The bacillus megaterium is applied to colonizing root system soil and has at least one of the functions of dissolving phosphorus, dissolving potassium, secreting IAA and the like, so that the growth of peanuts is promoted and the yield is increased. The bacillus amyloliquefaciens is applied to Solid-Surface motion (SSM) under the induction of sucrose and derivatives thereof, colonizes the root surfaces and roots under the condition of relatively low soil moisture content, and has a remarkable inhibiting effect on fusarium neosporae, a pathogenic bacterium of peanut rot, so that the plant immunity is improved, the microbial flora is adjusted, and the peanut rot is prevented and treated.

The third invention provides a formula and a preparation method of a biological bacterial fertilizer (a powder type special soil improvement and restoration agent for peanuts): (1) The bacillus mucilaginosus GF-32a, the bacillus megaterium GD-16 and the bacillus amyloliquefaciens GF-3 pure bacteria are subjected to liquid fermentation, and the diatomite is utilized to prepare the microbial inoculum. (2) 0.05-0.1 hundred million/g of paenibacillus mucilaginosus microbial inoculum, 0.2-0.5 hundred million/g of bacillus megaterium microbial inoculum, 0.5-1.0 hundred million/g of bacillus amyloliquefaciens microbial inoculum, 75.0-85.0 percent of solid fermentation products of livestock and poultry manure, 3.0-5.0 percent of sugarcane filter mud, 3.0-5.0 percent of humic acid, 8.0-10.0 percent of potassium sulfate, 3.0-5.0 percent of limestone powder and 1.0-3.0 percent of plant ash, which are mixed according to the proportion.

The fourth invention provides a combined application method of biological bacterial fertilizer (powder type soil improvement and restoration agent special for peanuts): the special soil improvement and restoration agent for 40-80 kg/mu peanuts is scattered on the surface of sandy soil or sandy loam (the water content is 15-20%) before sowing, the soil is deeply turned over by 25-30 cm, and 20-30 kg/mu inorganic compound fertilizer (N: P2O5: K2O = 15.

The application of the fertilizer is characterized in that the application is to prevent and treat peanut rot, promote growth by dissolving phosphorus, dissolving potassium, fixing nitrogen, helping beneficial bacteria colonize or secrete IAA and the like, degrade autotoxic substances of peanuts, and adjust microbial community structure and soil pH, so that pathogenic microorganisms are antagonized, the soil environment is improved, the plant immunity is improved, the disease index of the peanut rot in continuous cropping lands is effectively reduced, and the purposes of preventing and treating peanut soil-borne diseases and increasing yield are achieved.

In a specific embodiment, the application is to prevent and treat peanut rot, and the peanut rot can promote the growth of peanuts by dissolving phosphorus, dissolving potassium, fixing nitrogen, promoting beneficial bacteria to colonize or secrete IAA and the like, increase mineral elements, degrade autotoxic substances of the peanuts, adjust the microbial community structure and soil pH and the like, adjust the physical and chemical properties of soil, antagonize pathogenic microorganisms, improve the soil microenvironment, improve the plant immunity, effectively reduce the disease index of the peanut rot in continuous cropping land and achieve the purposes of preventing and treating peanut soil-borne diseases and increasing yield.

In a specific embodiment, one of the composition according to the second aspect of the present invention and the biological bacterial fertilizer (powder-type soil improvement and remediation agent for peanuts) according to the third aspect of the present invention is applied before the peanuts are sown.

Unless otherwise defined, all terms used in the present invention are commonly used in the art.

The method for improving and restoring soil by using the peanut rhizosphere microbial strain and the biological bacterial fertilizer thereof provided by the invention is described in detail below with reference to the examples, but the invention is not to be construed as being limited by the scope of the invention.

Example 1

Separation and purification of bacterial strains

10g of soil samples of 3 peanut rhizosphere soil samples of 3 natural villages, such as 3 farms of the scientific and technical faculty academy of north China in Changli county, qinhuang island city, hebei province, 90 mL of sterile water, three glass beads, 220 rpm shaking in a shaking table for 30min, standing for 30 sec, and preparing 10 ^-1 A soil dilution; using a pipette from 10 ^-1 Sucking 1mL of the soil diluent, adding into a large test tube containing 9mL of sterile water, and mixing to obtain 10 ^-2 A soil dilution; then from 10 ^-2 Sucking 1mL of the soil diluent, adding into a large test tube containing 9mL of sterile water, and mixing to obtain 10 ^-3 The soil diluent is analogized by analogy, 10 is respectively prepared ^-3 ，10 ^-4 ，10 ^-5 The soil dilution was followed by pipetting 100. Mu.l each of 10 dilutions ^-3 、10 ^-4 、10 ^-5 The diluted solution was passed through a silicate medium (formulation: sucrose 5.0g, agar 20.0g ₂ HPO ₄ 2.0g，MgSO ₄ 0.5g，CaCO ₃ 0.1g，FeCl ₃ 0.1g, distilled water to a constant volume of 1000.0mL, natural pH) was applied by a dilution application plate method, the sample at each concentration was repeated three times, and inverted culture was performed at 28 ℃ for 2 days. Selecting single colony, performing separation and purification culture by plate streaking method to obtain GF-32a, and storing with 20% glycerol at-20 deg.C for use.

The strains GD-16 and GF-3 are the strains stored in the laboratory, wherein the classification and identification of the strains GF-3 are detailed in the separation and identification of the peanut vein-invading red shell fungus fruit rot biocontrol bacillus and disease prevention effect.

EXAMPLE 2 identification of the strains

(1) For morphological identification, refer to "handbook of identification of common bacteria systems": GF-32a is gram-negative bacteria, and the bacterial colony is round and protuberant, such as a half glass ball; colorless and transparent, smooth surface, viscous, elastic, capable of being pulled into filamentous shape (figure 1), thick and long rod-shaped thallus, thick capsule, large spore, ellipse midlife, thallus size of 0.9-1.0 μm × 2.5-4.0 μm (figure 2). GD-16 is gram-positive bacterium, the colony is round, milky white, smooth in surface, sticky, easy to pick up, non-transparent, neat in edge, rod-shaped, multi-double-rod continuous, spore-producing, 1.32-1.70 x 3.73-6.60 μm.

GF-32a is activated on a silicate culture medium, and after GD-16 strains are activated on an LB culture medium, the physiological and biochemical characteristics of the GD-16 strains are analyzed and determined by a Meiriee VITEK-2 Compact full-automatic bacteria identification and analysis system and a biochemical identification card respectively.

TABLE 1 GF-32a Biochemical characterization

BXYL

+

AGAL

+

INO

+

dMNE

+

PVATE

-

NaCl6.5%

-

LysA

-

ALaA

(+)

MdG

+

dMLZ

+

AGLU

-

KAN

-

AspA

-

TyrA

+

ELLM

-

NAG

-

dTAG

-

OLD

-

LeuA

+

BNAG

+

MdX

-

PLE

+

dTRE

(-)

ESC

+

PheA

+

APPA

-

AMAN

+

IRHA

+

INU

+

TTZ

-

ProA

-

CDEX

-

MTE

+

BGLU

+

dGLU

+

POLYB_R

-

BGAL

+

dGAL

+

GlyA

-

BMAN

+

dRIB

(+)

PyrA

-

GLYG

(+)

dMAN

-

PHC

-

PSCNa

-

TABLE 2 GD-16 physiological and biochemical identification

AMY

-

AspA

-

PyrA

+

dRIB

-

dMAN

+

dTRE

+

PIPLC

-

BGAR

+

BGUR

-

ILATk

+

dMNE

-

ADH2s

-

dXYL

-

AMAN

-

AlaA

-

LAC

+

MBdG

-

OPTO

+

ADH1

+

PHOS

(-)

TyrA

+

NAG

+

PUL

-

BGAL

+

LeuA

-

dSOR

+

dMAL

+

dRAF

+

AGLU

(-)

ProA

-

URE

-

BACI

-

O129R

-

APPA

-

BGURr

-

POLYB

-

NOVO

-

SAL

+

CDEX

-

AGAL

+

dGAL

+

NC6.5

+

SAC

+

(3) 16S rDNA sequencing and sequence analysis

Extracting strain genome DNA by a boiling method, carrying out PCR amplification by taking 16S rDNA universal primers of 16SF (SEQ ID No. 3) and 16SR (SEQ ID No. 4) as templates, detecting an amplified product by using 1% agarose gel electrophoresis, cutting gel, recovering a target fragment, connecting the target fragment with a T-easy carrier, transforming the obtained product to E.coli JM109 competent cells after connection, sequentially adding 16 mul of IPTG solution, 40 mul of X-gal solution and 150 mul of bacterial liquid into an LB solid plate, uniformly coating the mixture, and culturing at 37 ℃ overnight. Screening blue white spots to select positive clones for PCR and electrophoresis detection. And (4) sending the bacterial liquid of the successfully detected positive clone to Shanghai biological engineering GmbH for sequencing. The sequence result is spliced by software DNAMAN 6.0 to obtain 16S rDNA sequences of 1501bp GF-32a and 1516bp GD-16 strains, and the sequences are shown as SEQ ID No.1 and SEQ ID No. 2. BLAST comparison analysis of the strain in GenBank shows that the 16S rDNA sequence similarity of the GF-32a strain and the Paenibacillus mucilaginosus (Paenibacillus mucolignosus) with the gene accession number AY646228 is 99.87 percent, and the 16S rDNA sequence similarity of the strain GD-16 and the Bacillus megaterium (gene accession numbers CP049296 and MN 240409) is 99.60 percent.

Based on the above results, GF-32a was designated as Paenibacillus mucilaginosus (GF-32 a) GF-32a (hereinafter, abbreviated as GF-32 a), and GD-16 was designated as Bacillus megaterium GD-16 (hereinafter, abbreviated as GD-16).

Example 3 preliminary verification of the ability to dissolve phosphorus, dissolve potassium and fix nitrogen

The bacterial solution of the GF-32a strain was cultured in a silicate medium (formula: sucrose 5.0g, na) by plating on a clean bench ₂ HPO ₄ 2.0g，MgSO ₄ 0.5g，CaCO ₃ 0.1g，0.5% FeCl ₃ ·H ₂ O1.0 mL, agar 20.0g, distilled water to 1000.0mL, pH7.2-7.4) and activating the GD-16 and GF-3 strains in LB medium (formula: yeast extract 5.0g, peptone 10.0g, naCl 10.0g, agar 20.0g, distilled water 1000.0mL, pH 7.0), beating a uniform-sized cake on the activated culture with a 1mL tip for a pipette, and then dividing the cake into individual portionsRespectively placing in potassium-resolving culture medium (GF-32 a culture medium formula is as follows, glucose 5g, ammonium sulfate 0.5g, yeast powder 0.5g, magnesium sulfate 0.3g, disodium hydrogen phosphate 2g, ferrous sulfate 0.03g, manganese sulfate 0.03g, potassium feldspar 2g, agar 20 g), potassium-resolving bacteria separation culture medium (GD-16 and GF-3 culture medium formula is as follows, na ₂ HPO ₄ 2.0g, sucrose 5.0g, mgSO ₄ •7H ₂ O 5.0mg，FeCl ₃ 5.0mg, calcium carbonate 1.0g, potassium feldspar powder 10.0g, distilled water 1000.0mL, agar 2.0g, pH7.3), and nitrogen-free medium (formula as follows, mannitol 10g, KH ₂ PO ₄ 0.20g，MgSO ₄ •7H ₂ O5.0 mg, sodium chloride 0.20g, caCO ₃ 5.0g，CaSO ₄ •2H ₂ 0.10g of O, 1000.0mL of distilled water, 20.0g of agar and 7.3 of pH, and an organophosphorus degrading bacterium isolation medium (the formula is as follows: (NH) ₄ ) ₂ SO ₄ 0.50g，MgSO ₄ 0.30g, 0.30g sodium chloride, 0.30g KCl, caCO ₃ 1.0g，FeSO ₄ 0.03g, lecithin 0.20g, agar 20.0g, glucose 10.0g, mnSO ₄ 0.03g, 1000.0mL of distilled water, pH 7.0) and an inorganic phosphate decomposing bacteria separation medium (the formula is as follows: (NH) ₄ ) ₂ SO ₄ 0.50g，MgSO ₄ 0.30g, 0.30g sodium chloride ₃ (PO ₄ ) ₂ 10.0g, 10.0g of glucose, mnSO ₄ 0.03g，FeSO ₄ 0.03g of agar, 20.0g of agar, 1000.0mL of distilled water and pH 7.2), and after 3 days, oil drop-shaped bacterial colonies can grow in a potassium-dissolving culture medium, hydrolysis rings can appear around an organophosphorus bacteria separating culture medium and an inorganic phosphorus bacteria separating culture medium, the diameters of the hydrolysis rings are 5mm and 5mm respectively, and the growth can be carried out on a nitrogen-free culture medium, which indicates that the culture medium has the capabilities of dissolving phosphorus, dissolving potassium and fixing nitrogen (figure 3), GD-16 has hydrolysis rings around the potassium-dissolving bacteria separating culture medium, the organophosphorus bacteria separating culture medium and the inorganic phosphorus bacteria separating culture medium, the diameter of the potassium-dissolving hydrolysis ring is 9mm, the diameter of the organophosphorus hydrolysis ring is 11mm, the diameter of the inorganic phosphorus hydrolysis ring is 11mm, and the culture medium can continue to grow on the nitrogen-free culture medium, and indicates that GD-16 has the capabilities of dissolving potassium, dissolving inorganic phosphorus, organic phosphorus and fixing nitrogen.

Example 4 determination of phosphorus solubilizing ability

Quantitative determination of phosphate-solubilizing capacity of the strain: activated GF-32A, GD-16 and GF-3 strains are inoculated in a PKO liquid culture medium (the formula is as follows: 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, 1000mL of distilled water and pH value adjusted to 7.0), shaking and culturing is carried out for 3d at 30 ℃, the centrifuged supernatant is collected, a molybdenum blue colorimetric method is used, 5 50mL colorimetric tubes are taken firstly and coded into five numbers of 1, 2, 4, 6 and 8, a phosphorus standard No.2 solution (a phosphorus standard solution: anhydrous potassium dihydrogen phosphate is weighed and taken out in 0.4391g of 1000mL of water as No.1 solution, phosphorus is contained in 0.1mg/mL of water, 10mL of the No.1 solution is absorbed, water is added to dilute to 100mL of phosphorus containing 0.01mg/mL of No.2 according to obtain a No.2 solution, and the colorimetric solution is added to water, and then the water is added to obtain 1, 2, 4, 6, 8, 9, 8 and 2mL of phosphorus containing 0.1, 8mL of water is added to obtain a 2mL of 2, 8mL of the colorimetric solution. Then 8.0mL of 0.015% hydrazine sulfate solution and 8.0mL of 2.5% sodium molybdate dilute sulfuric acid solution are added into each 5 tubes (140 mL of sulfuric acid is measured and injected into 300mL of water, the sulfuric acid is shaken up, the mixture is cooled to room temperature, 12.5g of sodium molybdate is added, the mixture is dissolved and added with water to 500mL, the mixture is shaken up and stands for 24 hours), 2.0mL of plug is added, the mixture is shaken up and removed, the 5 tubes are placed in a boiling water bath to be heated for 10min, the mixture is taken out and cooled to room temperature, the mixture is diluted to 50mL with water and fully shaken up, after 10min, the extinction value is respectively measured by a spectrophotometer under the wavelength of 650nm, a 1cm liquid bath and zero point adjustment by water. The extinction values were plotted on the ordinate and the phosphorus (0.01, 0.02, 0.04, 0.06, 0.08 mg) on the abscissa to obtain a standard curve. During color comparison, a pipette is used for sucking 10mL of a liquid to be detected, the liquid is injected into a 50mL colorimetric tube, 8.0mL of 0.015% hydrazine sulfate is added, 2.0mL of a sodium molybdate dilute sulfuric acid solution is added, a plug is added, the mixture is shaken up, the colorimetric tube is placed in a boiling water bath for heating for 10min, the mixture is taken out and cooled to room temperature, the mixture is diluted to 50mL by water and shaken up fully, after 10min, a spectrophotometer is used for 650nm, a 1cm liquid tank is used for adjusting the zero point by water, and the extinction value is measured. The content of available phosphorus in the supernatant was measured, and the results are shown in Table 3. The phosphorus dissolving efficiencies of GF-32a and GD-16 are 70.52 percent and 88.35 percent respectively.

TABLE 3 phosphorus solubilizing efficiency of the strains

Strain numbering	Phosphorus content (mg/l)	Phosphorus dissolution efficiency (%)
			GD-16	206.7122	88.35%
GF-32a	187.1507	70.52%
			CK	109.7506	--

EXAMPLE 5 measurement of Potassium-solubilizing ability

GF-32a, GD-16 and GF-3 bacterial suspensions were prepared in sterile deionized water, centrifuged and the supernatant (OD) was discarded ₆₀₀ About 0.5). Inoculating 5mL of bacterial suspension into 95mL of potassium-deficient medium (formula: 10.0 g/L sucrose, 2.0g/L Na) ₂ HPO ₄ ，1.0 g/L (NH ₄ ) ₂ SO ₄ ，0.5 g/L MgSO ₄ ·7H ₂ O,0.1 g/L NaCl,0.5 g/L yeast powder, 10.0 g/L potassium feldspar powder and pH7.2-7.4. Autoclaved at 115 ℃ for 30 min) in a 250mL Erlenmeyer flask, and shake-culturing at 28 ℃ and 180rpm/min for 7d with the same volume of sterile water added as a blank. The culture solution is subjected to H ₂ O ₂ After the treatment, the K + concentration in the solution was determined on a flame photometer. Adding 10mL of bacteria culture solution into 2mL 6%H ₂ O ₂ Digesting in boiling water bath for 1h, centrifuging at 8000rpm/min for 5min, collecting supernatant, and measuring K on flame photometer ^＋ And (4) concentration. The potassium standard solution is prepared by potassium-deficient culture medium without adding potassium feldspar powder.

1mol/L neutral NH ₄ OAc solution: weighing chemically pure CH ₃ COONH ₄ 77.09g of the mixture is diluted by water and the volume is nearly 1L. By HOAC or NH ₃ ·H ₂ O was adjusted to pH7.0 and then diluted to 1L.100 μ g/mL potassium standard stock solution: weighing KCl (analytically pure, dried at 110 deg.C for 2 h) 0.1907g in potassium-deficient culture medium without potassium feldspar powder or sterile deionized water or 1mol/L NH ₄ And (4) adding the solution to the OAc solution to a constant volume of 1L to obtain the standard stock solution containing 100 mu g/mL potassium. Potassium series standard solutions: accurately sucking 100 mug/mL potassium standard stock solutions of 0, 2.5, 5.0, 10.0, 15.0 and 20.0mL respectively, putting the stock solutions into a 100mL volumetric flask, and performing constant volume by using a potassium-deficiency culture medium without adding potassium feldspar powder to obtain potassium series standard solutions of 0, 2.5, 5.0, 10.0, 15.0 and 20.0 mug/mL.

The results show (Table 4) that both GF-32a and GD-16 have potassium-dissolving capacity, wherein the potassium-dissolving capacity of GF-32a is strongest, and the potassium-dissolving efficiency is as high as 84.37%.

TABLE 4 Potassium solubilizing efficiency of the strains

Strain numbering	Potassium content (μ g/mL)	Potassium decomposition efficiency (%)
			GD-16	8.94	48.10
GF-32a	11.13	84.37
			CK	6.04	--

EXAMPLE 6 Primary screening of strains for degradation of autotoxic substances

Performing experiment by agar block method, inoculating GF-32a on silicate culture medium agar plate with phenolic acid as unique carbon source at concentration of 100mg/L, and inoculating GD-16 and GF-3 on inorganic salt culture medium agar plate (formula is as follows: (NH) ₄ ) ₂ SO ₄ 2g，KH ₂ PO ₄ 2g，Na ₂ HPO ₄ 1.3g, 100mg of phenolic acid substances, 5g of NaCl, 1000mL of water and 6.8-7.2), culturing by taking different phenolic acids as unique carbon sources, and observing the growth condition of the strain after 48 h. The results show that the three strains can degrade phenolic acid substances, but GD-16 and GF-3 have weaker degradation capability, and GF-32a has stronger degradation capability (figure 4).

EXAMPLE 7 qualitative determination of IAA

Purified GF-32a, GD-16 and GF-3 strains were inoculated in a silicate liquid medium and LB liquid medium to which L-tryptophan (80 mg/L) was added, respectively, shake-cultured at 30 ℃ for 24 hours, and whether the strains secreted IAA was determined by Salkowski colorimetry. The operation method comprises the following steps: 50 μ L of the suspension was dropped onto a white ceramic plate, and an equal volume of Salkowski colorimetric solution (50mL 35% HCl lO) was added ₄ +1mL 0.5mol/L FeCl ₃ ) And a mixed solution of 50. Mu.L of an LB liquid medium to which no inoculum is added and an equal volume of a colorimetric solution is used as a control. And observing the white ceramic plate after the white ceramic plate is placed at room temperature in a dark place for 30min, wherein the condition that the color changes into pink is positive and indicates that the IAA can be secreted, and the darker the color indicates that the secretion intensity is higher, the condition that the color does not change into negative and indicates that the IAA cannot be secreted. As a result, none of the blank control, GF-32a, and GF-3 was discolored, indicating that the blank control was negative and did not secrete IAA. GD-16 changed to pink, indicating that it was positive for its ability to secrete IAA, and was able toPromoting the growth of peanut plants.

Example 8 determination of optimum pH

GF-32a, GD-16 and GF-32a single colonies are respectively picked and activated in a liquid silicate culture medium and an LB liquid culture medium, GF-32a is inoculated into 10mL of silicate liquid culture medium with different pH values according to the inoculation amount of 5 percent, GD-16 and GF-3 are inoculated into 10mL of LB liquid culture medium with different pH values according to the inoculation amount of 5 percent, and the pH values of the culture media are respectively 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0. Each process set 3 replicates. Performing shake culture at 30 deg.C and 200rpm/min, performing GF-32a shake culture for 24 hr, adding 1mL of bacterial suspension into 9mL of sterile water, performing serial gradient dilution, selecting 10 ^-3 ，10 ^-4 ，10 ^-5 Gradient sample coating, 30 ℃ culture for 48h counting. OD determination after 6h shaking culture of GD-16 and GF-3 ₆₀₀ . The results are shown in tables 5 and 6, the optimum pH value of GF-32a is between 6.0 and 7.0, the optimum pH value of GD-16 is about 7.0, and the optimum pH value of GF-3 is about 6.0, which shows that the optimum pH values of the three strains are weak acidity or neutrality, and provides theoretical basis for the formula development of the soil remediation agent special for peanuts and the colonization of probiotics in the soil of the peanut rhizosphere.

TABLE 5 enumeration of GF-32a shake flask fermentations at different pH's (48 h)

pH	GF-32a（10 ⁵ cfu/mL）
		3.0	0.00±0.00c
4.0	0.00±0.00c
		5.0	109.00±39.34b
6.0	211.33±60.80a
		7.0	242.67±70.44a
8.0	1.33±1.53c
		9.0	2.00±0.00c
10.0	0.00±0.00c

TABLE 6 OD600 measurements (48 h) for GD-16 and GF-3 shake flask fermentations at different pH' s

pH	GD-16	GF-3
			3.0	0.1070±0.0030f	0.0687±0.0055e
4.0	0.1717±0.0035e	0.0743±0.0021ef
			5.0	2.1440±0.0520c	0.49307±0.0260c
6.0	2.6640±0.0655b	1.4873±0.0738a
			7.0	3.0013±0.0446a	1.3213±0.0480b
8.0	0.5773±0.0085c	0.2503±0.0218d
			9.0	0.5017±0.0635d	0.0113±0.0038f
10.0	0.1063±0.0070f	0.0330±0.0066ef

Example 9 carbohydrate-induced solid surface movement (SSM movement)

The preparation of a medium in which sucrose in a silicate medium was replaced with glucose, fructose, maltose and lactose, respectively, after GF-32a was activated on a silicate solid medium, the medium was inoculated into the center of the plate of the prepared silicate medium, and the medium was incubated at 30 ℃ for 24 hours using a sucrose-containing silicate medium as a blank control, and as a result, it was found that glucose, fructose, maltose, lactose and sucrose had no effect on the movement of Paenibacillus mucilaginosus on the solid surface (Table 7). Respectively replacing glucose in a PDA culture medium with sucrose, fructose, maltose and lactose to prepare a culture medium, respectively adding glucose, fructose, maltose, sucrose and lactose to prepare a culture medium in an LB culture medium, activating GD-16 and GF-3 on the LB solid culture medium, inoculating GF-32a to the centers of flat plates of the prepared PDA culture medium and LB culture medium, and culturing at 30 ℃ for 24h, wherein the results show (Table 8) that glucose and sucrose can induce the movement capacity of GD-16 on the solid surface and fructose, lactose and sucrose can induce the movement capacity of GF-3 on the solid surface in the LB culture medium compared with a blank control; in PDA medium, glucose, fructose and maltose have better effect on GD-16 diffusion, and glucose and sucrose have better effect on GF-3 diffusion. In conclusion, the sucrose and the glucose can induce the movement capability of GD-16 and GF-3 on the solid surface, and are beneficial to colonizing on the surface of the peanut root to form a bacterial film on the surface of the root system. The method provides theoretical basis for the colonization of the soil at the rhizosphere and the surface of the peanut by the probiotics and the formula development of the soil remediation agent special for the peanut.

TABLE 7 Effect of different saccharides on GF-32a diffusion

	GF-32a colony diameter (cm)
		Silicate medium (sucrose)	0.73±0.08a
Silicate medium (glucose)	0.73±0.09a
		Silicate medium (fructose)	0.67±0.10a
Silicate medium (maltose)	0.91±0.24a
		Silicate medium (lactose)	0.78±0.01a

TABLE 8 Effect of the diffusion of different saccharides GD-16 and GF-3

	GD-16 colony diameter (cm)	GF-3 colony diameter (cm)
			LB	1.19±0.04c	0.73±0.063c
LB + glucose	1.83±0.53a	1.83±0.18c
			LB + fructose	1.47±0.08bc	8.05±1.34a
LB + maltose	1.55±0.20abc	1.03±0.11c
			LB + lactose	1.29±0.29bc	4.21±1.32b
LB + sucrose	1.76±0.22abc	2.35±0.21c
			PDA (glucose)	1.45±0.15a	2.40±0.21a
PDA (fructose)	1.46±0.04a	1.40±0.36b
			PDA (maltose)	1.48±0.08a	1.20±0.28b
PDA (lactose)	1.13±0.16b	0.83±0.05b
			PDA (sucrose)	1.22±0.029b	2.42±0.60a

Example 10 CaCO ₃ Promotion of GF-32a growth

The activated GF-32a colony is picked up in 5mL of silicate liquid culture medium, and the mixture is shaken overnight at 30 ℃ and 200rpm to prepare seed liquid. Inoculating the seed liquid into CaCO with different contents according to the inoculation amount of 5 percent ₃ Silicate solution ofIn a culture medium, shaking culture is carried out for 3d at 30 ℃ and 200rpm/min, plate colony counting is carried out, and the pH value of fermentation liquor is measured. Adding 100 μ L of shake-cultured fermentation broth into 900 μ L of sterile deionized water, purging, mixing, and performing serial gradient dilution to obtain 10 ^-3 、10 ^-4 And 10 ^-5 Diluting, spreading 100 μ L of the diluted solution on silicate culture medium, culturing at 30 deg.C for 48 hr, and counting. Each dilution was performed in 3 replicates. The results show that CaCO3 (Table 9) is effective in balancing acids generated during GF-32a growth and metabolism, and the largest amount of GF-32a cells in the fermentation broth is obtained at a concentration of 0.3-0.5g/L, while adjusting pH. The method provides theoretical basis for formula development of the soil remediation agent special for peanuts and colonization of the peanut rhizosphere soil by probiotics.

TABLE 9 different contents of CaCO ₃ Effect on GF-32a growth amount (72 h)

CaCO ₃ Concentration (g/L)	Cell concentration (10) ⁶ cfu/mL）
		0	73.8571±21.5053d
0.1	185.1429±26.0923b
		0.2	199.4286±32.5415b
0.3	408.2857±82.6856a
		0.4	367.8571±20.9398a
0.5	370.8571±31.4612a
		0.8	121.8571±21.8512c
1.6	158.7143±21.4065bc

EXAMPLE 11 liquid fermentation of probiotics

Streaking strain GF-32a glycerol strain on a silicate solid culture medium, streaking strain GD-16 and GF-3 glycerol strain on an LB solid culture medium, and culturing at 30 ℃ for 48h to obtain activated bacteria; the activated bacteria are transferred into a seed liquid culture medium (namely a silicate bacteria liquid culture medium and an LB liquid culture medium), and cultured for 12 hours at 30 ℃ and 200rpm to obtain a first culture which is used as a seed liquid.

Transferring the first culture as seed liquid of liquid shake flask fermentation to a liquid fermentation medium (containing Paenibacillus mucilaginosus GF-32a with a formula of molasses 2.0%, soybean meal 1.0%, and MgSO 2% ₄ ·7H ₂ 0 0.2％、K ₂ HPO ₄ 0.08％、CaCO ₃ 0.1 percent, and the fermentation medium formula of the Bacillus megaterium GD-16 is as follows: corn starch 1.0%, bean flour 1.0%, NH ₄ ) ₂ SO ₄ 0.5%、MgSO ₄ ·7H ₂ O 0.1%、FeSO ₄ ·7H ₂ O 0.03%、K ₂ HPO ₄ ·3H ₂ O 0.02%、KH ₂ PO ₄ 0.01 percent, and the formula of the Bacillus amyloliquefaciens GF-3 fermentation medium is as follows: corn starch 1.00%, bean flour 1.00%, (NH) ₄ ) ₂ SO ₄ 0.50%、MgSO ₄ ·7H ₂ O 0.10%、FeSO ₄ ·7H ₂ O 0.03%、K ₂ HPO ₄ ·3H ₂ O 0.02%、KH ₂ PO ₄ 0.01%、ZnSO ₄ ·7H ₂ O 0.04%、CaCO ₃ 0.04%、MnSO ₄ ·H ₂ O0.02%) was cultured at 200rpm at 30 ℃ for 72 hours in a flask to obtain a second culture. The second culture was bathed at 85 ℃ for 15min to kill the trophozoites, then diluted in a gradient and selected to have a concentration of 10 ^-4 、10 ^-5 、10 ^-6 The bacterial suspension is plated and counted, each treatment is repeated for 3 times, after 30 ℃ inversion culture for 24h (GF-32 a inversion culture for 48 h), the colony number of each plate is selected to be between 30 and 300 for plate counting, and the plate colony counting result is respectively GF-32a:4.13 +/-0.07 x 10 ⁸ Per mL, GD-16:3.36 +/-0.03 multiplied by 10 ⁹ seed/mL, GF-3: 1.08. + -. 0.06X 10 ¹⁰ One per mL.

Example 12 preparation of biological bacterial manure (powdery peanut-dedicated soil improvement remediation agent)

And (3) centrifugally collecting the second culture, adding a protective agent, and then spraying and drying to prepare GF-32a, GD-16 and GF-3 microbial inoculum for later use.

Respectively mixing 0.2-0.5 hundred million/g of paenibacillus mucilaginosus microbial inoculum, 0.2-0.5 hundred million/g of bacillus megaterium microbial inoculum and 0.5-1.0 hundred million/g of bacillus amyloliquefaciens microbial inoculum with organic fertilizers (75.0-85.0% of livestock and poultry manure solid fermentation product, 3.0-5.0% of sugarcane filter mud, 3.0-5.0% of humic acid and 5.0-20.0% of soil) to respectively prepare a GF-32a biological organic fertilizer, a GD-16 biological organic fertilizer and a GF-3 biological organic fertilizer for later use.

The compound microbial fertilizer is prepared by mixing 0.05-0.1 hundred million/g of paenibacillus mucilaginosus microbial inoculum, 0.2-0.5 hundred million/g of bacillus megaterium microbial inoculum and 0.5-1.0 hundred million/g of bacillus amyloliquefaciens microbial inoculum with organic fertilizer (75.0-85.0% of livestock and poultry manure solid fermentation product, 3.0-5.0% of sugarcane filter mud, 3.0-5.0% of humic acid and 5.0-20.0% of soil) for later use.

Mixing 0.05-0.1 hundred million/g paenibacillus mucilaginosus agent, 0.2-0.5 hundred million/g bacillus megaterium agent, 0.5-1.0 hundred million/g bacillus amyloliquefaciens agent, 75.0-85.0% of solid livestock and poultry manure fermentation product, 3.0-5.0% of sugarcane filter mud, 3.0-5.0% of humic acid, 8.0-10.0% of potassium sulfate, 3.0-5.0% of limestone powder and 1.0-3.0% of plant ash according to the proportion to prepare the biological bacterial fertilizer (the powder type soil improvement repairing agent special for peanuts) for later use.

Example 13 potted plant experiments to verify growth promoting effects

A Jihua No. 16 peanut pot culture experiment is carried out in a greenhouse, 1kg of sterilized soil is filled in each pot, the sterilized soil and 5g of microbial inoculum are mixed uniformly, and watering is carried out regularly after sowing. The treatment is respectively 0.1 hundred million/g paenibacillus mucilaginosus GF-32a microbial inoculum, 0.2 hundred million/g bacillus megaterium GD-16 microbial inoculum, 0.5 hundred million/g bacillus amyloliquefaciens GF-3 microbial inoculum and three complex microbial inoculants (0.1 hundred million/g GF-32a, 0.2 hundred million/g GD-16 and 0.5 hundred million/g GF-3), and blank controls are set. Each treatment was repeated 3 times, 40 pots were repeated, the main root length was counted at 0d,7d,14d and 21d when the first pair of true leaves appeared after the peanut seedlings came out of the soil, and the growth promoting ability of each treatment was examined.

As can be seen from table 10, the length of the main root of the treatment inoculated with the composite strain is longer than that of the control, which is beneficial to the peanuts to better absorb water and nutrients in the soil and to the growth of the peanuts compared with the treatment inoculated with the single strain.

TABLE 10 Effect of the strains on peanut plants

Example 14 greenhouse plot experiment verification of growth promotion and production increase effects

A plot experiment is carried out in a greenhouse of a Changli school district of the university institute of science and technology in Hebei to verify the growth promoting and yield increasing effects of the soil improvement repairing agent special for peanuts.

The soil type is sandy loam, crops are not planted in previous stubbles, the peanut variety is Jihua No. 16, 50 kg/mu of biological bacterial fertilizer (powder type special soil improvement and repair agent for peanuts) is applied to a treatment group, deep ploughing is carried out for 20cm, each treatment is repeated for 3 times, and each repetition is 20m ² . The bio-organic fertilizer is a blank control. And (5) counting the emergence situation and the yield per mu, and calculating the emergence rate and the yield increase rate.

The results show (table 11): the germination rate of the biological bacterial manure (powder type peanut special soil improvement repairing agent) is 96.67%, the yield per mu is 341.29kg, the yield increase rate is 11.96%, the germination rates of the GF-32a biological organic fertilizer and the GD-16 biological organic fertilizer are 91.83% and 91.33%, the yield per mu is 330.52kg and 327.09kg, and the germination rate and the yield per mu are obviously higher than those of the conventional control. The result shows that the GF-32a and GD-16 biological organic fertilizer has a remarkable promotion effect on the growth of peanuts.

TABLE 11 peanut emergence and yield enhancement

Treatment of	Rate of emergence (%)	Mu yield (kg/mu)	Yield increase (%)
				GF-32a biological organic fertilizer	91.83±1.04b	330.52±16.28b	8.42±0.73b
GD-16 biological organic fertilizer	91.33±1.26b	327.09±15.45c	7.30±0.47c
				GF-3 biological organic fertilizer	87.33±4.01bc	306.70±13.30d	0.63±0.31d
Biological bacterial fertilizer (soil special for peanuts)Soil improvement repairing agent	96.67±0.76a	341.29±16.96a	11.96±0.77a
				Blank control	84.33±3.01c	304.79±13.07d	0.00e

Example 15 field disease prevention growth promotion test

Disease prevention and growth promotion tests of biological bacterial fertilizer (powder type special soil improvement repairing agent for peanuts) and a matching method thereof are respectively carried out in a Huzhuanzhuan test field (the soil type of the test field is sand, the fertility level before sowing is shown in table 12, and the continuous cropping is 3 years) in Hebei province Qianhan city and Huzhuangan new house village test field (the soil type of the test field is sand, the fertility level before sowing is shown in table 12, and the fertility level before sowing is 2 years) in Hebei province Qianhan city. The peanut variety is Jihua No. 16, and all treated peanut seeds including blank control are dressed by using a suspension seed coating agent (3% fludioxonil +3% thifluzamide) and dried in the shade for later use.

The treatment dosage is as follows: 50 kg/mu of GF-32a biological organic fertilizer, 50 kg/mu of GD-16 biological organic fertilizer, 50 kg/mu of GF-3 biological organic fertilizer, 50 kg/mu of biological bacterial fertilizer (powder type special soil improvement repairing agent for peanuts), 50 kg/mu of compound microbial fertilizer, 50 kg/mu of organic fertilizer and 50 kg/mu of inorganic fertilizer (8.0-10.0 percent of potassium sulfate, 3.0-5.0 percent of limestone powder, 1.0-3.0 percent of plant ash and 82.0-88 percent of soil) are taken as conventional controls, and the treatment without fertilizer application before sowing is a blank control.

Before sowing, when the relative water content of sandy soil or sandy loam is 65-70% (water content is 15-20%), the treated fertilizers are spread on the soil surface, turning is carried out for 25-30 cm, and when sowing is carried out, 25 kg/mu of inorganic compound fertilizer (total nutrient is more than or equal to 45%, N-P) is applied in ditches ₂ O ₅ -K ₂ O: 15-15-15) and covering with film and sowing according to the density of 10000-12000 holes/mu. Each treatment is repeated for 3 times, and the peanut fruit rot disease is graded and investigated by a five-point sampling method 10 days before harvest, and the yield and the control effect on the peanut fruit rot are measured. Pod grading criteria were as follows: level 0: the pods are intact and have no rotting symptoms; stage 1: the pericarp has infection spots, and the fruit is intact; and 2, stage: 1/2 of pod rot; and 3, level: rot 1/2 of the pod. Disease index, prevention and treatment efficiency, dry fruit acre yield and yield increase rate are respectively expressed by formulas (1), (2), (3) and (4).

Disease index = ∑ (rotten fruit x the rotten fruit level)/(total fruit number x highest level) x 100

Formula (1)

Control efficiency (%) = (disease index in blank control area-disease index in treatment area)/disease index in blank control area x 100

Formula (2)

Dry fruit per mu yield = wet fruit per mu yield × 50% × 85%

Formula (3)

Yield increase (%) = (treatment zone per mu yield-blank control zone per mu yield)/blank control zone per mu yield x 100

Formula (4)

Disease prevention in the field experiments showed (table 14): the seed dressing Jihua No. 16 (3% fludioxonil +3% thifluzamide) with the suspended seed coating agent still has peanut rot, the disease index of the field in one continuous cropping year is 36.49 +/-3.82, the peanut rot is more serious when the number of the continuous cropping years is larger, the disease index of the field in two continuous cropping years is 33.46 +/-4.15, and the field control effect of chemical agent treatment (blank control) on the peanut rot is very limited.

In a land parcel with 3 years of continuous cropping (Table 13), the control efficiency of the GF-32a bio-organic fertilizer, the GD-16 bio-organic fertilizer and the GF-3 bio-organic fertilizer on peanut fruit rot is 52.78% + -2.84%, 48.62% + -7.70% and 50.34% + -3.01% respectively, the yield increase rate is 17.63% + -5.68%, 12.97% + -5.88% and 14.37% + -6.70% respectively, and the control efficiency is obviously higher than that of a blank control and the organic fertilizer. The control efficiency and the yield increase rate of the compound microbial fertilizer are respectively 65.37 +/-2.21% and 22.52 +/-7.20%, and are obviously higher than that of the single application of the biological organic fertilizer. The control efficiency and the yield increase rate of the inorganic fertilizer on peanut fruit rot are respectively 54.64% +/-8.04% and 15.43% +/-3.49%, and the control efficiency and the yield increase rate are between single application of the biological organic fertilizer and the biological bacterial fertilizer (powder type special soil improvement repairing agent for peanuts), and are not obviously different from the single application of the biological organic fertilizer and the biological bacterial fertilizer. The prevention and treatment efficiency and the yield increase rate of the biological bacterial manure (powder type special soil improvement and restoration agent for peanuts) on peanut rot are remarkably higher than those of other treatments, are respectively 90.50% +/-1.15% and 41.39% +/-3.11%, and the effect is relatively stable.

In a land parcel with 2 years of continuous cropping (Table 14), the control efficiency of the peanut rot by independently applying the XJ-32 bio-organic fertilizer, the GF-32a bio-organic fertilizer and the GF-3 bio-organic fertilizer is 56.31% + -4.22%, 57.49% + -2.79% and 58.70% + -3.82%, the yield increasing rate is 14.72% + -2.73%, 17.50% + -4.75% and 16.37% + -4.44%, and the control efficiency and yield increasing rate difference between the bio-organic fertilizers is smaller but is obviously higher than that of a blank control and the organic fertilizers. The control efficiency and the yield increase rate of the compound microbial fertilizer are respectively 70.93 percent and 26.22 percent, which are obviously higher than those of the single application of the biological organic fertilizer. The prevention and control efficiency and the yield increase rate of the inorganic fertilizer on the peanut fruit rot are respectively 61.52% +/-4.76% and 18.57% +/-3.71%, which are slightly better than the biological organic fertilizer and lower than the composite microbial fertilizer. The prevention and treatment effect and the yield increase effect of the biological bacterial manure (the powder type special soil improvement and restoration agent for peanuts) on peanut rot are obviously higher than those of other treatments, and the prevention and treatment effect and the yield increase effect are respectively as high as 96.23% +/-1.78% and 42.34% +/-5.63%.

In summary, the following steps: the microbial-organic-inorganic compound biological bacterial fertilizer (the powdery special soil improvement repairing agent for peanuts) is remarkable in prevention and treatment effect and yield increase effect, and the microbial fertilizer, the organic fertilizer and the inorganic fertilizer are independently applied, so that the synergistic effect of the biological bacterial fertilizer (the powdery special soil improvement repairing agent for peanuts) on the microbial fertilizer, the organic fertilizer and the inorganic fertilizer is shown.

TABLE 12 test soil fertility levels in the test fields

Site	pH	Quick-acting nitrogen (mg kg) ^-1 ）	Quick-acting phosphorus (mg.kg) ^-1 ）	Quick-acting potassium (mg.kg) ^-1 ）
					Buckle village town	5.81±0.18	135.63±8.53	38.72±9.26	102.37±17.52
Deng New house village	6.92±0.14	140.95±16.48	42.15±13.84	98.75±19.05

Control effect of table 13 deduction village and town test field

Treatment of	Index of disease condition	Control effect (%)	Mu yield (kg/mu)	Yield increase (%)
					Biological bacterial fertilizer (soil improvement repairing agent special for peanut)	3.18±0.39e	90.50±1.15a	355.95±7.83a	41.39±3.11a
Composite microbial fertilizer	11.59±0.74d	65.37±2.21b	308.45±18.12b	22.52±7.20b
					GF-3 biological organic fertilizer	16.62±1.01c	50.34±3.01c	287.93±17.61cd	14.37±6.70cd
GD-16 biological organic fertilizer	17.19±2.58c	48.62±7.70c	284.42±14.80de	12.97±5.88de
					GF-32a biological organic fertilizer	15.80±0.95c	52.78±2.84c	296.14±14.30c	17.63±5.68c
Organic fertilizer	27.32±5.09b	18.34±15.20d	277.00±21.26e	10.03±8.45e
					Inorganic fertilizer	15.18±2.69cd	54.64±8.04bc	290.60±8.79cd	15.43±3.49cd
Blank control	33.46±4.15a	0.00e	251.76±6.93f	0.00f

Control effect of surface 14 Deng New House village test field

Treatment of	Index of disease condition	Control effect (%)	Mu yield (kg/mu)	Yield increase (%)
					Biological bacterial fertilizer (soil improvement repairing agent special for peanut)	1.38±0.65e	96.23±1.78a	340.81±13.48a	42.34±5.63a
Composite microbial fertilizer	10.61±1.62d	70.93±4.45b	295.55±15.15b	26.22±3.81b
					GF-3 biological organic fertilizer	15.07±1.40c	58.70±3.82c	278.64±10.63c	16.37±4.44c
GF-32a biological organic fertilizer	15.51±1.02c	57.49±2.79c	281.33±11.37c	17.50±4.75c
					XJ-32 biological organic fertilizer	15.94±1.54c	56.31±4.22c	274.70±6.54cd	14.72±2.73cd
Organic fertilizer	30.84±3.80b	15.48±10.44d	262.97±9.26d	9.83±3.87d
					Inorganic fertilizer	14.04±1.74cd	61.52±4.76bc	283.91±8.89c	18.57±3.71bc
Blank control	36.49±3.82a	0.00e	239.44±3.34e	0.00e

Example 16 microbial community structural changes

Peanut rhizosphere soil treated by a special soil improvement remediation agent (Qa 8) for peanuts in the test field of New Deng House village and treated by a blank Control (CK) is collected by a five-point sampling method and sent to Baimaike Biotech limited to carry out microbial diversity analysis by a double-End sequencing method (Paired-End) based on an Illumina HiSeq sequencing platform. And performing species annotation and abundance analysis by performing mosaic filtering, clustering or denoising on the Reads. The results show (FIG. 5)&6): fusarium in rhizosphere soil of blank Control (CK),StagonosporopsisThe abundance of sp, et al pathogenic fungi was 8.15% and 7.84%, respectively, accounting for about 15.99%. And Fusarium in rhizosphere soil treated by Qa8,StagonosporopsisThe abundance of sp, et al pathogenic fungi was reduced to 1.55% and 2.15%, respectively, accounting for about 3.7%, indicating that Qa8 treatment could significantly reduce the abundance of pathogenic microorganisms.

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 amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Sequence listing

<110> 11 Hubei institute of science and technology

<120> method for improving and repairing soil by using peanut rhizosphere microbial strain and biological bacterial fertilizer thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1501

<212> DNA

<213> Paenibacillus mucilaginosus (Paenibacillus mucinarginosus)

<400> 1

agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60

ggagcacttc ggtgcttagc ggcggacggg tgagtaacac gtaggcaacc tgcctgtaag 120

atcgggataa ctaccggaaa cggtagctaa gaccggatgg ctggtttcgg tgcatgccgg 180

aatcatgaaa cacggggcaa cctgtggctt acggatgggc ctgcggcgca ttagctagtt 240

ggcggggtaa tggcccacca aggcgacgat gcgtagccga cctgagaggg tgatcggcca 300

cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga atcttccgca 360

atgggcgcaa gcctgacgga gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag 420

ctctgttgcc agggaagaat gtcgtggaga gtaactgctc tgcgaatgac ggtacctgag 480

aagaaagccc cggctaacta cgtgccagca gccgcggtaa tacgtagggg gcaagcgttg 540

tccggaatta ttgggcgtaa agcgcgcgca ggcggtcttt taagtctggt gtttaagccc 600

ggggctcaac cccggttcgc accggaaact ggaagacttg agtgcaggag aggaaagcgg 660

aattccacgt gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcgg 720

ctttctggac tgtaactgac gctgaggcgc taaagcgtgg ggagcaaaca ggattagata 780

ccctggtagt ccacgccgta aacgatgagt gctaggtgtt aggggtttcg atacccttgg 840

tgccgaagta aacacaataa gcactccgcc tggggagtac gctcgcaaga gtgaaactca 900

aaggaattga cggggacccg cacaagcagt ggagtatgtg gtttaattcg aagcaacgcg 960

aagaacctta ccaggtcttg acatccctct gaaagcccta gagatagggc cctccttcgg 1020

gacagaggtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080

gtcccgcaac gagcgcaacc cttgacttta gttgccagca ttgagttggg cactctagag 1140

tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200

gacctgggct acacacgtac tacaatggcc ggtacaacgg gaagcgaagt cgcgagatgg 1260

agcgaatcct tagaagccgg tctcagttcg gattgcaggc tgcaactcgc ctgcatgaag 1320

tcggaattgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggtcttgta 1380

cacaccgccc gtcacaccac gagagtttac aacacccgaa gccggtgggg taacccgtaa 1440

gggagccagc cgtcgaaggt ggggtagatg attggggtga agtcgtaaca aggtaaccgt 1500

a 1501

<210> 2

<211> 1516

<212> DNA

<213> Bacillus megaterium (Bacillus megaterium)

<400> 2

agagtttgat cctggctcag gatgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60

gaactgatta gaagcttgct tctatgacgc tagcggcgga cgggtgagta acacgtgggc 120

aacctacctg taagactggg ataacttcgg gaaaccgaag ctaataccgg ataggatctt 180

ctccttcatg ggagatgatt gaaagatggt ttcggctatc acttacagat gggcccgcgg 240

tgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag ccgacctgag 300

agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta 360

gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggct 420

ttcgggtcgt aaaactctgt tgttagggaa gaacaagtac aagagtaacc gcttgtacct 480

tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 540

ggtggcaagc gttatccgga attattgggc gtaaagcgcg cgcaggcggt ttcttaagtc 600

tgatgtgaaa gcccacggct caaccgtgga gggtcattgg aaactgggga acttgagtgc 660

agaagagaaa agcggaattc cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac 720

cagtggcgaa ggcggctttt tggtctgtaa ctgacgctga ggcgcgaaag cgtggggagc 780

aaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa gtgttagagg 840

gtttccgccc tttagtgctg cagctaacgc attaagcact ccgcctgggg agtacggtcg 900

caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 960

attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaac tctagagata 1020

gagcgttccc cttcggggga cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc 1080

gcgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt gccagcattt 1140

agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1200

atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggatggt acaaagggct 1260

gcaagaccgc gaggtcaagc caatcccata aaaccattct cagttcggat tgtaggctgc 1320

aactcgccta catgaagctg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata 1380

cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac acccgaagtc 1440

ggtggagtaa ccgtaaggag ctagccgcct aaggtgggac agatgattgg ggtgaagtcg 1500

taacaaggta accgta 1516

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agagtttgat cctggctcag 20

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tacggttacc ttgttacgac tt 22

Claims

1. Paenibacillus mucilaginosus (Paenibacillus mucinicus) GF-32a, wherein the strain GF-32a is preserved with the preservation number of CGMCC No.22902.

2. The strain GF-32a of claim 1, wherein the 16S rDNA sequence of the strain GF-32a is shown in SEQ ID NO. 1.

3. An improved microbial strain for peanuts in a continuous cropping land, which comprises the strain GF-32a of claim 1 or 2, bacillus megaterium GD-16 and Bacillus amyloliquefaciens GF-3, wherein the preservation number of the Bacillus megaterium GD-16 is CGMCC No.24218.

4. The microbial inoculum prepared by the microbial strains according to claim 3, wherein the viable count ratio of the strains GF-32a, GD-16 and GF-3 is 0.05-0.1 hundred million/g: 0.2-0.5 hundred million/g: 0.5-1.0 hundred million/g.

5. The biological bacterial fertilizer prepared by the microbial inoculum according to claim 4, which is characterized by further comprising the following raw materials in percentage by mass: 75.0 to 85.0 percent of livestock and poultry excrement solid fermentation, 3.0 to 5.0 percent of sugarcane filter mud, 3.0 to 5.0 percent of humic acid, 8.0 to 10.0 percent of potassium sulfate, 3.0 to 5.0 percent of limestone powder and 1.0 to 3.0 percent of plant ash.

6. Use of the strain GF-32a according to claim 1 or 2 or the microbial strain according to claim 3 or the microbial inoculum according to claim 4 or the biological bacterial fertilizer according to claim 5 for improving and remediating peanut soil.

7. A method for improving and repairing continuous cropping peanut soil is characterized by comprising the following steps: before peanut sowing, the microbial strain of claim 3, the microbial inoculum of claim 4 or the biological bacterial fertilizer of claim 5 is broadcast to a sowing field.

8. The method according to claim 7, wherein the biological bacterial manure is applied at a rate of 40 to 80kg/acre.

9. The method according to claim 7 or 8, characterized by further comprising 20-30kg/mu of inorganic compound fertilizer in the spreading process.

10. The method according to claim 9, characterized in that the step of further comprising, after the step of broadcasting, turning the microbial fertilizer into soil by a deep ploughing depth of 25 to 30cm, and furrow-applying the inorganic compound fertilizer during sowing.