CN110628674B - Bacillus pumilus with functions of improving acid soil and removing potassium and preparation and application of microbial inoculum thereof - Google Patents
Bacillus pumilus with functions of improving acid soil and removing potassium and preparation and application of microbial inoculum thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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Abstract
The invention discloses a bacillus pumilus with functions of improving acid soil and removing potassium and preparation and application of a microbial inoculum thereof; according to the thallus morphology, the colony characteristics and the physiological and biochemical characteristics of the HG-8 strain, the strain is identified as bacillus pumilus by combining the analysis results of 16S rDNA and gyrB sequences, and is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 9 and 26 months with the preservation number of CGMCC NO. 18620. The HG-8 strain can be stably colonized in acid soil; the microbial inoculum prepared by the strain can be applied to improving the growth of crops such as wheat, corn, hot pepper and the like in acid soil; the pH value of the acid soil and the content of effective potassium can be obviously improved, and the crop yield is increased; the strain has salt tolerance and excellent fermentation performance, and the microbial inoculum has simple preparation process, short fermentation period and low cost, and is beneficial to industrial production and transportation.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to bacillus pumilus with functions of improving acid soil and removing potassium, and preparation and application of a microbial inoculum thereof.
Background
Soil acidification refers to the phenomenon that soil acidity becomes strong under natural and artificial conditions, namely the pH value of soil is reduced, and the essence of the soil is that salt-based cations in the soil are leached out, and exchangeable acids are increased, so that the pH value of the soil is reduced, and the soil acidification is one of important manifestations of soil degradation. Soil acidification causes the reduction of soil nutrient availability, the enhancement of heavy metal ion activity, soil hardening, few gaps and poor air permeability, is not beneficial to the regulation of water, air, heat and fertilizer in soil, causes soil degradation, further influences the normal growth and development of crops, and finally causes the reduction of the yield and the quality of the crops.
From the 20 th century and 80 th century to the present, the condition of soil acidification in China is more and more serious, and the acidified land area is expanded to more than 40% of the cultivated land area. The main reason for affecting soil acidification is affected by human industrial activities and agricultural activities, the main form being acid rain and excessive application of chemical fertilizers.
At present, the treatment measures for soil acidification mainly comprise modes of acid rain control, biological improvement, agricultural measures, soil conditioner use and the like. Common soil conditioners include lime conditioners, minerals and industrial wastes, microbial fertilizers, biomass charcoal, organic materials and the like.
Potassium is one of three essential nutrients for crop growth and development, and is involved in plant photosynthesis, protein synthesis, carbohydrate metabolism, enzyme activation and other metabolism, so that the stress resistance of plant can be improved and the quality of crop can be improved. Potassium deficiency affects plant growth, root development, stress resistance and pest and disease resistance, resulting in low crop yield. At present, about 60 percent of cultivated land in China is lack of potassium, but silicate minerals in soil are rich, potassium element is difficult to release due to the fact that the silicate minerals are insoluble, in order to relieve the situation that the soil is lack of potassium, potassium is supplemented by chemical fertilizers, which is a method commonly used in agricultural production, but the soil structure is damaged due to the fact that the chemical fertilizers are excessively used for a long time, and nutrient loss and environmental pollution are caused; moreover, the crops can leave acid radical ions such as sulfate radicals and chloride ions after absorbing potassium ions in the fertilizer, and the acid radical ions further increase the acidification degree of the soil.
The potassium-dissolving bacteria can decompose silicate minerals, destroy the lattice structure of potassium feldspar, convert insoluble potassium in the minerals into soluble potassium, further increase the supply of quick-acting potassium in soil, promote the growth and development of crops, and improve the yield and quality of the crops. Many potassium-solubilizing bacteria have been reported to promote the growth of crops by promoting the absorption of phosphorus and potassium by plants through the ability of secreting organic acids (CN 105925497A). The separation and screening of the microbial strains which have the potassium-dissolving function and can improve the pH value of the soil are applied to the acid soil, so that the improvement of the acid soil and the growth promotion of crops are facilitated.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a bacillus pumilus HG-8 strain. The bacillus pumilus HG-8 is separated from wheat rhizosphere soil in saline-alkali soil of yellow river delta, has strong salt tolerance and excellent fermentation performance, can obviously improve the pH value of acid soil, and also has the potassium-dissolving function and the capability of promoting the growth of crops. Is a strain with excellent performance and wide application prospect.
Specifically, the invention relates to the following technical scheme:
the bacillus pumilus HG-8 provided by the invention is preserved in China general microbiological culture Collection center (CGMCC for short, and the address is No. 3 Xilu No.1 Beijing Kogyo-Yang district, Beijing) in 2019, 9 and 26 days, and the biological preservation number is as follows: CGMCC NO. 18620.
The thallus morphology and the colony characteristics of the bacillus pumilus HG-8 are as follows:
the colonies on the PDA plate are light yellow, round and translucent, the edges are neat, the surface is wet, the middle of the colonies is slightly raised, and the shape of the thalli observed under a microscope is rod-shaped.
The physiological and biochemical characteristics of the bacillus pumilus HG-8 are as follows:
the strain has the advantages that the oxygen demand is positive in facultative anaerobism, gram reaction, glucose oxidation fermentation test, catalase reaction, starch hydrolysis test, methyl red test, salt tolerance test, phenylalanine ammonia deaminase test and citrate utilization test; the V-P test and the nitrate reduction reaction are negative; can grow in LB liquid culture medium with NaCl concentration of 2% and 5%.
The bacillus pumilus HG-8 has the molecular biological characteristics that:
the 16S rDNA sequence of the strain is shown in SEQ ID NO.1, the gyrB sequence is shown in SEQ ID NO.2, and the rep-PCR gene fingerprint is shown in figure 3.
The microbial inoculum containing the bacillus pumilus HG-8 also belongs to the protection scope of the invention.
The microbial inoculum not only contains the bacillus pumilus HG-8, but also can comprise auxiliary materials or carrier matrixes and the like, such as trehalose, fulvic acid, turf, animal manure, straws of various crops, peanut shells, chemical fertilizers and the like.
In the microbial inoculum, the Bacillus pumilus HG-8 can exist in the form of cultured living cells or fermentation liquor of the living cells.
The formulation of the microbial inoculum can be liquid, emulsion, suspending agent, granule, powder, wettable powder or water dispersant.
Further, the invention also provides a preparation method of the bacillus pumilus HG-8 microbial inoculum, which comprises the following steps:
inoculating the seed liquid of the bacillus pumilus HG-8 into a fermentation culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: culturing at the rotation speed of 120r/min, the temperature of 30-32 ℃ and the tank pressure of 0.05-0.06 MPa for 20-30 h to obtain a bacillus pumilus HG-8 fermentation liquid;
the fermentation medium comprises the following components: soluble starch 12.5g, peptone 9.50g, K2HPO4 0.40g，MgSO40.5g and 1.00L of water.
Preferably, the inoculation amount of the seed solution of the Bacillus pumilus HG-8 is 8-12% (volume ratio).
Preferably, the preparation of the seed solution of Bacillus pumilus HG-8 comprises: activating strains, preparing a first-stage seed liquid and preparing a second-stage seed liquid; wherein:
the bacteria are activated as follows: streaking the bacillus pumilus HG-8 stored at low temperature on a LB solid culture medium plate, and culturing at 28-32 ℃ for 24-36 h; and (3) selecting a single colony of the HG-8 strain, streaking again, transferring to an LB solid culture medium plate, and culturing at 28-32 ℃ for 24-36 h for later use.
The first-stage seed liquid is prepared by the following steps: and inoculating the activated HG-8 strain lawn in an LB liquid culture medium, performing shake culture at the temperature of 28-32 ℃ for 12-16 h at 200-220 r/min to obtain a first-level seed solution.
The second-stage seed liquid is prepared by the following steps: inoculating the primary seed liquid into a secondary seed culture medium according to the volume percentage of 1-2% for fermentation culture, wherein the fermentation culture conditions are as follows: culturing at the rotation speed of 120r/min, the temperature of 30-32 ℃ and the tank pressure of 0.05-0.06 MPa for 12-16 h to serve as secondary seed liquid;
the formula of the secondary seed culture medium is as follows: soluble starch 12.5g, peptone 9.50g, K2HPO4 0.40g，MgSO40.5g and 1.00L of water.
In the preparation method, the prepared Bacillus pumilus HG-8 fermentation liquor can be directly used as a liquid microbial inoculum; further, the liquid microbial inoculum can be prepared into a solid microbial inoculum, and the preparation method comprises the following steps:
centrifuging the Bacillus pumilus HG-8 fermentation liquor at 6000-8000 r/min for 20-30 min, and collecting precipitates; and adding diatomite or corn flour into the precipitate to serve as a carrier matrix, wherein the addition amount of the carrier matrix is 6-8% of the weight of the Bacillus pumilus HG-8 fermentation liquor, and uniformly stirring and drying to obtain the solid microbial inoculum of the Bacillus pumilus HG-8.
The application method of the prepared bacillus pumilus HG-8 microbial inoculum comprises the following steps: uniformly mixing the bacillus pumilus HG-8 liquid fungicide with water according to a volume ratio of 1:200 (according to a dosage of 1.5-2.5L/mu) when in use, and irrigating roots; the solid microbial inoculum is used according to the dosage of 0.5-1.0 kg/mu, mixed with seeds or diluted by adding a proper amount of water and applied by irrigating roots; the two microbial agents can be used as strains of biological organic fertilizers and compound microbial fertilizers.
The application of the bacillus pumilus HG-8 or the microbial inoculum containing the bacillus pumilus HG-8 in at least one of the following 1) to 4) also belongs to the protection scope of the invention;
1) the pH value of the acid soil is increased, and the acid soil is improved;
2) promoting the growth of crops under the condition of acid soil;
3) converting the insoluble potassium to soluble potassium;
4) preparing the biological organic fertilizer and/or the compound microbial fertilizer.
Preferably, the crop is wheat.
The invention has the beneficial effects that:
(1) the bacillus pumilus HG-8 has strong potassium-dissolving capacity, the potassium-dissolving capacity of the bacillus pumilus HG-8 is determined according to a method specified in NY/T1847-2010, and the content of the effective potassium in a culture solution is increased by 57.94% in comparison with that of an uninoculated strain by inoculating an HG-8 strain in the culture solution containing potassium feldspar.
(2) The bacillus pumilus HG-8 disclosed by the invention can be stably colonized in acid soil, the pH value of the soil can be obviously increased, the soil environment is improved, and the growth of crops is promoted.
(3) The bacillus pumilus HG-8 has good fermentation property, simple microbial inoculum production process, short production period and low raw material cost, and is beneficial to industrial production and transportation.
(4) The Bacillus pumilus HG-8 strain has extremely high salt tolerance, can tolerate the salt concentration of 350g/L, can grow vigorously in a liquid LB culture medium with the salt concentration of 50-150 g/L, can grow well in a liquid LB culture medium with the salt concentration of 200-300 g/L, and can still grow in a liquid LB culture medium with the salt concentration of 350 g/L. The bacillus pumilus HG-8 strain can stably survive when coexisting with a fertilizer, and can be applied together with a water-soluble fertilizer or a solid-solution fertilizer.
Drawings
FIG. 1: a phylogenetic tree was constructed from the 16S rDNA sequence of HG-8 strain by multiple sequence homology analysis using the Alignment Explorer program in Mega 5.0 software.
FIG. 2: the constructed phylogenetic tree was analyzed by Mega 5.0 based on the gyrB sequence of HG-8 strain.
FIG. 3: HG-8 strain rep-PCR gene fingerprint.
FIG. 4: the potassium-dissolving capacity of HG-8 strain is determined by a standard curve, a linear regression equation is that y is 0.1521x-0.0042, and a coefficient R is determined2＝0.9998。
FIG. 5: HG-8 strain growth curves.
FIG. 6: and (5) primary screening results of the culture medium.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, although the soil contains abundant silicate minerals, the soil is difficult to release potassium because of its insolubility, which causes potassium deficiency. In order to relieve the situation of potassium deficiency of soil, a large amount of potassium fertilizer is generally applied at present, but acid radical ions such as sulfate radicals, chloride ions and the like are left after crops absorb potassium ions in the fertilizer, and the ions can increase the acidification degree of the soil.
The potassium-dissolving bacteria can be used for converting insoluble potassium in minerals into soluble potassium, but the potassium-dissolving bacteria reported in the prior art generally promote phosphorus and potassium absorption of plants by secreting organic acid, so that the application of the microbial strains which have the potassium-dissolving function and can improve the pH value of soil is more beneficial to the improvement of acid soil and the promotion of crop growth.
According to the invention, a salt-tolerant Bacillus pumilus (Bacillus pumilus) HG-8 is obtained by separating and screening wheat rhizosphere soil from saline-alkali soil of yellow river delta, and further research shows that the Bacillus pumilus (Bacillus pumilus) HG-8 has the following remarkable properties:
(1) has potassium-dissolving capacity and promotes the growth of crops;
(2) can be stably colonized in acid soil, remarkably improve the pH value of the soil and improve the soil environment;
(3) has excellent fermentation property, and can be fermented and cultured in a short time to obtain 109The number of the bacteria above CFU/mL;
(4) has extremely high salt tolerance and can tolerate the salt concentration of 350 g/L.
Therefore, the Bacillus pumilus (HG-8) is different from the currently reported Bacillus pumilus, integrates various excellent properties, and particularly can remarkably improve the pH value of soil; the extremely high salt resistance of the strain is beneficial to simultaneously applying the microbial inoculum produced by the strain and chemical fertilizer.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the experimental conditions not specified in the examples are specified, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, and the like used in the following examples are commercially available unless otherwise specified.
Example 1: isolation and selection of strains
Collecting wheat rhizosphere soil of yellow river delta saline-alkali soilSampling, taking 10g of soil sample under aseptic condition, dissolving in 90mL of sterile distilled water at a speed of 220r/min, performing shake culture for 30min, sucking 1mL of sterile distilled water and mixing with 9mL of sterile distilled water to obtain 10-11mL of the diluted solution of (1) to 10-1The diluted solution is mixed with 9mL of sterile water to obtain 10-2The dilution of (1) is prepared by analogy to dilution, and 10-3，10-4，10-5，10-6，10-7And a series of dilutions. The selected dilution is 10-5、10-6、10-7The soil dilution is respectively absorbed by 100uL and coated in a PDA culture medium, and the soil dilution is cultured for 1-3 d at the constant temperature of 30-32 ℃. Picking single colony, streaking to obtain pure culture, and storing.
And (3) performing a potassium-dissolving test on the obtained pure cultures, measuring the size of a potassium feldspar hydrolysis ring, determining the potassium-dissolving activity of the strains according to NY/882-.
PDA medium composition: 200g of potato, 5.0g of beef extract, 20g of glucose, 15g of agar, (NH)4)2SO4 1.0g，MgSO4 1.0g，KH2PO4 0.6g，CaCO33.0g, pH 6.8-7.2, 1L deionized water.
Potassium-dissolving medium components: sucrose 10.0g, (NH)4)2SO4 1.0g，MgSO40.5g, yeast extract 0.5g, Na2HPO42.0g，CaCO31.0g, potassium feldspar powder 1.0g, agar 15.0g and deionized water 1L.
Activating a plurality of obtained silicate bacteria, respectively scraping two rings of activated pure culture under aseptic condition, inoculating the pure culture into a seed liquid culture medium (LB liquid culture medium), performing shake culture at 30 ℃ for 8-10 h, inoculating the pure culture into the LB liquid culture medium with an inoculation amount of 2%, inoculating sterile water with the same amount into a blank control LB liquid culture medium, repeating three treatments, performing shake culture at 30 ℃ and 200r/min for 48h, measuring the pH value of fermentation liquor by using a precise pH test paper, fermenting the strains with the pH value of more than 8 for 48h by adopting the culture method, precisely measuring the pH value of the fermentation liquor by using a pH meter, and further screening the strains with high pH value of the fermentation liquor. And finally, screening to obtain the HG-8 strain which has high potassium-dissolving activity and can obviously improve the pH value of the fermentation liquor.
The seed liquid culture medium (LB liquid culture medium) comprises the following components: 5.0g of yeast powder, 10.0g of peptone, 10.0g of NaCl and 1L of distilled water.
The LB solid medium comprises the following components: 5.0g of yeast powder, 10.0g of peptone, 10.0g of NaCl and 15.0g of agar. 1L of distilled water.
Example 2: identification of strains
(1) Morphology of thallus and colony
The colony of the HG-8 strain on the PDA plate is light yellow, round and translucent, the edge is neat, the surface is wet, the middle of the colony is slightly raised, and the shape of the thallus observed under a microscope is rod-shaped.
(2) Physiological and biochemical characteristics
The physiological and biochemical characteristics of the HG-8 strain are shown in Table 1. The strain has the advantages that the oxygen demand is facultative anaerobic, gram reaction, glucose oxidation fermentation test, catalase reaction, starch hydrolysis test, methyl red test, phenylalanine ammonia-lyase test and citrate utilization test are positive; the V-P test and the nitrate reduction reaction are negative; can grow in liquid LB liquid culture medium with NaCl concentration of 2% and 5%. LB liquid medium composition: 10g of peptone, 5g of yeast powder, 10g of NaCl and 1L of water.
Table 1: physiological and biochemical characteristics of HG-8 strains
Note: + is positive; negative.
(3) Sequence analysis of 16S rDNA of strain
The 16S rDNA sequence of the HG-8 strain is shown in SEQ ID NO. 1; the sequence is compared with the sequence in a Genbank database by Blast analysis, the homology of HG-8 and the strain of bacillus is found to be higher, 8 strains with higher sequence similarity with HG-8 strains are selected for phylogenetic analysis, and the phylogenetic tree is constructed by utilizing the Alignment Explorer program in Mega 5.0 software for multi-sequence homology analysis (figure 1). The 16S rDNA sequence of the HG-8 strain has homology of 99% with the publicly published 16S rDNA of the Bacillus pumilus (GQ 281299), and the HG-8 strain is identified as the Bacillus pumilus by combining the determination results of the thallus morphology, the colony characteristics and the physiological and biochemical indexes of the HG-8 strain.
(4) gyrB sequence analysis of HG-8 Strain
The gyrB sequence of the HG-8 strain is shown as SEQ ID NO. 2; performing Blast analysis comparison on the sequence and a sequence in a Genbank database, finding that strains with higher homology belong to the genus Bacillus, selecting 18 strains with higher sequence similarity with HG-8 strains for phylogenetic analysis, and constructing a phylogenetic tree based on gyrB complete sequence by using a Neighbor-Joining method by using Mega 5.0 software (figure 2). The homology of the gyrB sequence of the HG-8 strain and the publicly published 16S rDNA of Bacillus pumilus (CP029464) reaches 99%, and the HG-8 strain is further identified to be Bacillus pumilus (Bacillus pumilus) HG-8 by combining the 16S rDNA identification result, the thallus morphology, the colony characteristics and the physiological and biochemical characteristics.
Based on the morphological identification and molecular identification results of the strains, the HG-8 strain obtained by separation is identified as Bacillus pumilus (Bacillus pumilus) and named as Bacillus pumilus (Bacillus pumilus) HG-8. And the strain is subjected to biological preservation, and the preservation information is as follows:
the strain name is as follows: bacillus pumilus
Latin name: bacillus pumilus
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 9/26/2019
Registration number of the preservation center: CGMCC NO. 18620.
Example 3: HG-8 strain rep-PCR gene fingerprint.
1. HG-8 strain genome DNA extraction
The TIANGEN bacterial genomic DNA extraction kit DP302 was used.
(1) 1mL of HG-8 strain culture solution is taken, centrifuged at 10000rpm for 1min, and supernatant is completely sucked as much as possible;
(2) adding 110 μ L TE buffer solution and 70 μ L lysozyme into the thallus precipitate, and treating at 37 deg.C for more than 30 min;
(3) adding 20 mu L of protease K solution into the tube, and uniformly mixing;
(4) adding 200 μ L buffer solution GB, shaking for 15sec, standing at 70 deg.C for 10min, cleaning solution, and centrifuging for 10-15sec to remove water droplet on the inner wall of the tube cover;
(5) adding 220 μ L of anhydrous ethanol, shaking thoroughly, mixing for 15sec, wherein flocculent precipitate may appear, and centrifuging for 10-15sec to remove water drop on the inner wall of the tube cover;
(6) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12000rpm for 30sec, pouring off waste liquid, and placing an adsorption column CB3 into the collecting pipe;
(7) adding 500 μ L buffer GD (before use, whether absolute ethyl alcohol is added or not is checked), centrifuging at 12000rpm for 30sec, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
(8) adding 600 μ L of rinsing solution PW (checking whether anhydrous ethanol is added before use) into adsorption column CB3, centrifuging at 12000rpm for 30sec, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
(9) repeating the operation step 8;
(10) the adsorption column CB3 was put back into the collection tube, centrifuged at 12000rpm for 2min, and the waste liquid was discarded. The adsorption column CB3 is placed at room temperature for a plurality of minutes until the residual rinsing liquid in the adsorption material is completely dried.
(11) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50 mu L of eluent TE into the middle part of the adsorption film, standing at room temperature for 3min, centrifuging at 12000rpm for 2min, and collecting the solution into the centrifuge tube. The solution collected by the centrifugal tube is the HG-8 strain genome DNA.
2. Rep-PCR gene fingerprint of HG-8 strain
The invention carries out Box-PCR on HG-8 strain genome DNA by using a BOX-AIR primer. HG-8 strain Box-PCR primer sequences are shown in Table 2.
Table 2: HG-8 strain Box-PCR primer sequence
The BOX-AIR primer amplification system (50. mu.L) is: 10 XTaq PCR master mix 25. mu.L, template 1. mu.L, BOX-AIR 2. mu.L, with ddH2The content of O is filled to 50 mu L. The rep-PCR reaction conditions were as follows: 7min at 95 ℃; 1min at 94 ℃, 1min at 53 ℃, 8min at 65 ℃ and 35 cycles; 16min at 65 ℃; pause at 4 ℃.
HG-8 strain Box-PCR product is subjected to 1% agarose gel electrophoresis, the electrolyte is 1 × TAE, Trans2K DNA Marker and NormalRunTM 250bp-IV DNA ladder are selected as the markers, and electrophoresis is carried out for about 20min at constant voltage of 220V.
FIG. 3 is a gene fingerprint of Box-PCR products of genomic DNA of HG-8 strain and 7 strains of Bacillus pumilus stored in this laboratory. Through electrophoretic strip contrastive analysis, the number of the HG-8 strain repetitive sequence units and the characteristics of the repetitive sequences on chromosome distribution are found, the repetitive sequences have differences between species levels and different strains of the same species, and the electrophoretic patterns are completely consistent through repeated tests and have good repeatability. Therefore, the method can be used for identifying and identifying the strains in patent strain HG-8 dimensional right.
16S rDNA and gyrB gene identification and gene fingerprint information of the HG-8 strain provide basis for identifying and identifying the strain in patent strain Bacillus pumilus HG-8 right.
Example 4: potassium-solubilizing ability assay of HG-8 Strain
The potassium-dissolving capacity of the HG-8 strain is determined by a method specified in NY/T1847-2010.
Inoculating the HG-8 strain seed liquid culture medium into a potassium dissolving culture medium according to the proportion of 2 percent (volume ratio) to be used as a test group, adding sterile deionized water according to the proportion of 2 percent (volume ratio) to be used as a blank control, and culturing for 7d under the conditions of 28 ℃ and 200r/min of a shaker. The treatment group and the blank control group are respectively provided with 5 repetitions, the relative error of the obtained results does not exceed 10%, and the measurement results are expressed by an arithmetic mean value.
The seed culture solution comprises the following components: 10g of peptone, 5g of yeast powder, 10g of NaCl and 1L of water.
And (3) decomposing potassium fermentation liquid culture medium: sucrose 10.0g, (NH)4)2SO4 1.0g，MgSO40.5g, yeast extract 0.5g, Na2HPO42.0g，CaCO31.0g, 1.0g of potassium feldspar powder and 1.0L of deionized water.
Transferring the whole fermentation liquid into digestion tube, digesting and concentrating to about 10mL, adding 2mLH2O2Continuing to evaporate the solution, and repeatedly adding 30% H2O2The solution is applied several times until the viscous material is completely digested. Taking down and cooling, centrifuging at 3500r/min for 10min, collecting the supernatant in a 50mL volumetric flask, adding water to a constant volume of 50mL, and then measuring the content of the quick-acting potassium in the solution.
Preparation of standard solution: sucking potassium standard solution 0mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL and 6.00mL, placing in 8 volumetric flasks respectively, adding 10.00mL nitric acid solution, and fixing the volume with water. This solution was a standard series of solutions containing potassium in an amount of 0. mu.g, 1.00. mu.g, 2.00. mu.g, 3.00. mu.g, 4.00. mu.g, 5.00. mu.g, and 6.00. mu.g in 1 mL. Adjusting the zero point of the instrument on a flame photometer by using a blank solution, adjusting the full scale of the instrument by using a standard solution with the highest concentration in the standard solution series, measuring other standard solutions, and recording the indication value of the instrument. A calibration curve was plotted or a linear regression equation was calculated from the potassium concentration and the instrument indication (FIG. 4).
And (4) determining the content of the effective potassium in the culture medium of the test group and the culture medium of the blank control group by using the standard curve. According to the requirement that the increase of quick-acting potassium in the culture solution containing potassium feldspar is increased by more than 20% compared with the increase of non-acting potassium specified in NY/T1847-2010, the content of quick-acting potassium in the culture solution of the HG-8 strain is increased by 57.94% compared with the control, and is 2.9 times of that in the NY/T1847-2010 standard.
Example 5: salt tolerance of HG-8 Strain
1. Salt tolerance of HG-8 Strain
(1) Salt resistance test
Inoculating liquid LB culture medium fermentation liquor (with a spore rate of more than 95%) of HG-8 strain into liquid LB culture media with NaCl concentrations of 50g/L, 100g/L, 150g/L, 200g/L, 250g/L, 300g/L and 350g/L respectively according to the inoculation amount of 2% (volume ratio), performing shake flask culture for 24 hours at 32 ℃ at 200-220 r/min. The number of HG-8 strains in liquid LB medium with different NaCl concentrations was determined by gradient dilution and plate coating counting. Setting three repeats
The results show that the HG-8 strain has extremely high salt tolerance, can tolerate the salt concentration of 350g/L, can grow vigorously in a liquid LB culture medium with the salt concentration of 50-150 g/L, can grow well in a liquid LB culture medium with the salt concentration of 200-300 g/L, can still grow in a liquid LB culture medium with the salt concentration of 350g/L, and the salt tolerance test results are shown in Table 3.
Table 3: growth conditions of HG-8 strains in liquid LB culture media with different NaCI concentrations
Note: + + +, vigorous growth; + + good growth; and + can grow.
2. Survival rate test of HG-8 strain in water-soluble fertilizer
(1) Survival rate of HG-8 bacterial strain in liquid water-soluble fertilizer
The test selects the liquid water-soluble fertilizer product of Shandong agriculture fertilizer industry science and technology limited company, and the main components of the product are as follows: macroelements are more than or equal to 400g/L, total nitrogen is more than or equal to 360g, potassium is more than or equal to 45g/L, humic acid is more than or equal to 30g/L, nitrate nitrogen is more than or equal to 90g/L, ammonium nitrogen is more than or equal to 90g/L, and amide nitrogen is more than or equal to 180 g/L.
Adding 10% (volume ratio) of HG-8 strain liquid LB culture medium fermentation liquor (with a spore rate of more than 95%) into a liquid water-soluble fertilizer, uniformly mixing, sealing and storing in a shade, measuring the number of thalli in the water-soluble fertilizer every 30 days, and continuously measuring for 180 days. The result shows that the thallus quantity of the HG-8 strain in the liquid water-soluble fertilizer is not greatly reduced, the overall level is higher, and the loss rate is lower. After 180 days, the survival rate of the thallus is 95.92%, and the test results are shown in Table 4.
Table 4: thallus number change of HG-8 bacterial strain in liquid water-soluble fertilizer
(2) Survival rate of HG-8 bacterial strain in solid water-soluble fertilizer
The test selects a solid water-soluble fertilizer (ammonium nitrate yellow potassium-humic acid-containing water-soluble fertilizer) product of Shandong agriculture and big fertilizer industry science and technology Limited, and the product mainly comprises the following components: N-P2O5-K217-5-23 percent of O, more than or equal to 45 percent of macroelements, more than or equal to 9 percent of nitrate nitrogen and more than or equal to 3 percent of fulvic acid.
Adding HG-8 strain fermentation liquor (with a spore rate of more than 95%) into a solid water-soluble fertilizer according to a volume ratio of 10%, uniformly mixing, placing the mixture in a super-clean workbench to dry for 6 hours under an aseptic condition, sealing and storing the mixture in a cool place, measuring the number of thalli in the water-soluble fertilizer every 30 days, and continuously measuring for 180 days. The results show that the change trend of the number of thalli in the solid water-soluble fertilizer is stable, the overall level is high, the loss rate of the thalli is low, the survival rate of the thalli is 87.58% after 180 days, and the test results are shown in Table 5.
Table 5: thallus quantity change of HG-8 bacterial strain in solid water-soluble fertilizer
The liquid LB medium components involved in the above experiments: 10g of peptone, 5g of yeast powder, 10g of NaCl and 1L of water.
Example 6: determination of colonization ability of HG-8 strain in plant rhizosphere
(1) Screening and stability detection of HG-8 strain double-antibiotic mutant strain
Scraping 2-ring activated HG-8 strain lawn in LB liquid culture medium without rifampicin, culturing at 32 ℃ to logarithmic phase, inoculating seed liquid into LB liquid culture medium with rifampicin concentration of 0.5. mu.g/mL in an inoculum size of 2% (volume ratio), culturing for 8-24 h, inoculating the culture liquid into LB liquid culture medium with rifampicin concentration of 1. mu.g/mL in an inoculum size of 2% (volume ratio), culturing for 24h, and sequentially inoculating into LB liquid culture medium with rifampicin concentration of 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL, 200. mu.g/mL, 250. mu.g/mL, 300. mu.g/mL. Inoculating the obtained rifampicin-resistant strain into LB liquid culture medium containing 300 mug/mL rifampicin and 5 mug/mL spectinomycin (Spe), screening the dual-resistant strain simultaneously resisting rifampicin and spectinomycin, wherein the concentration of rifampicin in LB liquid culture medium is 300 mug/mL, the antibiotic concentration of the anti-spectinomycin strain and the antibiotic concentration of the anti-rifampicin strain are the same, repeating the activation for 3 generations on a plate without rifampicin and spectinomycin, and then respectively returning to the LB liquid culture medium containing 300 mug/mL rifampicin and 300 mug/mL spectinomycin for culture, finally obtaining the dual-resistant marker strain which can be inherited stably and can resist rifampicin and spectinomycin simultaneously.
(2) Determination of colonization ability of HG-8 strain in wheat rhizosphere
HG-8 double-antibiotic mutant strains obtained in the above tests are inoculated into LB culture medium without antibiotics and subjected to shake culture at 28 ℃ overnight. 1mL of the bacterial suspension was transferred to LB medium containing rifampicin (300. mu.g/mL) and spectinomycin (300. mu.g/mL) and shake-cultured at 28 ℃ for 12 hours. Then taking 1mL of well-grown bacterial liquid to transfer to LB liquid culture medium without antibiotics, culturing for 48h to obtain double-antibiotic labeled bacterial liquid, centrifuging the bacterial liquid to remove supernatant to prepare bacterial suspension, and adjusting the concentration of the bacterial to 108CFU/mL。
And (2) respectively filling acid soil with the pH value of 5.35 into flowerpots with the inner diameter of 22cm, sowing wheat seeds, and selecting 20 pots with consistent wheat growth vigor when wheat seedlings are about 5-8 cm high, wherein 10 pots are a treatment group for applying the double-antibody marked HG-8 strain, and 10 pots are a control group for applying the inactivated double-antibody marked HG-8 strain. 15mL of double-antibody-labeled HG-8 strain bacterial suspension is irrigated into each basin of the treatment group, and 15mL of inactivated double-antibody-labeled HG-8 strain bacterial suspension is irrigated into each basin of the control group.
Recovering rhizosphere soil of wheat at 10, 20, 30 and 40 days after root irrigation, coating on double antibiotic flat plate by gradient dilution coating method, and countingAnd detecting the colonization condition of the HG-8 strain at the rhizosphere of the wheat. The test results are shown in table 6. With the extension of the test time, the number of the treated HG-8 double-resistant labeled strains in the rhizosphere of the wheat is reduced to a certain degree, but the number of the strains in each gram of dry soil at 40d still reaches 4.5 multiplied by 105CFU/g, while no colonies were produced in the control group, indicating that HG-8 strain was able to stably colonize the rhizosphere of wheat.
Liquid LB medium composition: 10.0g of peptone, 5.0g of yeast powder, 10.0g of NaCl and 1.0L of water.
Solid LB medium composition: 10.0g of peptone, 5.0g of yeast powder, 10.0g of NaCl, 15.0g of agar and 1.0L of water.
Table 6: colonization result of HG-8 strain in wheat rhizosphere
Example 7: influence of double-antibody-labeled HG-8 strain on pH value of rhizosphere soil of wheat
And (2) respectively filling acid soil with the pH value of 5.35 into flowerpots with the inner diameter of 22cm, sowing wheat seeds, and selecting 20 pots with consistent wheat growth vigor when wheat seedlings are about 5-8 cm high, wherein 10 pots are a treatment group for applying the double-antibody marked HG-8 strain, and 10 pots are a control group for applying the inactivated double-antibody marked HG-8 strain. 15mL of double-antibody-labeled HG-8 strain bacterial suspension is irrigated into each basin of the treatment group, and 15mL of inactivated double-antibody-labeled HG-8 strain bacterial suspension is irrigated into each basin of the control group.
Collecting wheat rhizosphere soil samples at 10 th, 20 th, 30 th, 40 th, 50 th and 60 th days after root irrigation. Spreading rhizosphere soil on clean newspaper, spreading into thin layer, and air drying in shady and ventilated place. When the soil sample reaches a semi-dry state, the large soil blocks are crushed in time, so that the sample completely passes through a test sieve with the aperture of 1 mm. By potentiometric method to be CO-free2Water as extractant, 7g ± 0.1g of sieved soil sample was weighed into a 15mL centrifuge tube and mixed at a ratio of 1: 2.5 to 7mL of carbon dioxide-free water (deionized water is injected into a conical flask, boiled for 10min, sealed and cooled for standby). Sealing the container, shaking for 5min, standing for 1 hr, and measuring each soil with pH meterThe pH of the sample. ANOVA analysis is carried out on the pH values of rhizosphere soil of the blank group and different strains by using SAS 9.0 data analysis software, and the pH value of the rhizosphere soil of the HG-8 strain is significantly different from that of the blank control (P) according to the last sampling of 60d<0.05), indicating that the HG-8 strain can obviously improve the pH value of the rhizosphere soil of the wheat. The measurement results are shown in Table 7.
Table 7: influence result of HG-8 strain on pH value of wheat rhizosphere soil
Note: and (4) carrying out statistical analysis on the pH values of rhizosphere soil samples of different strains. Different letters (a, b) indicate significant differences between the different samples.
Example 8: optimization of HG-8 strain thallus fermentation medium formula
(1) Determination of seed age
HG-8 strain seed liquid is inoculated into 12 bottles of sterile LB seed liquid culture medium prepared in the same batch in equal amount according to the inoculation amount of 2 percent (volume ratio), and shake culture is carried out at 32 ℃ and under the condition of 200 r/min. The OD of the seed solution was measured at a wavelength of 600nm from the initial inoculation time, every 1 hour for 12 hours in a row, and 3 replicates each time. The growth curve of the strain was plotted as arithmetic mean values from the OD values of the seed liquid at each time period measured at a wavelength of 600nm (fig. 5). As can be seen from FIG. 5, the log phase of the HG-8 strain is 3-9 h, the stationary phase is 10h later, and the fermentation time of the seed liquid is determined to be 9 h.
(2) Screening of basal fermentation Medium
Taking 33 Bacillus pumilus spore fermentation formulas obtained from consulted literature as basic test culture medium of HG-8 strain, counting colonies of each fermentation culture medium by gradient dilution coating method when spore rate reaches above 90%, wherein the formula with the best fermentation character is No. 22 formula, fermentation time is 42h, spore rate is above 99%, and thallus number is 2.7 × 109cfu/mL. The test results are shown in Table 8 and FIG. 6.
33 basal medium components:
medium No. 1: 5.0g of soluble starch, 20.0g of peptone, 1.0g of monopotassium phosphate, 0.5g of ammonium sulfate and 1.0L of water.
Medium No. 2: 10.0g of glucose, 20.0g of corn flour, 20.0g of soybean cake powder, 6.0g of corn steep liquor, 1.0g of sodium chloride, 0.2g of manganese sulfate and 1.0L of water.
Medium No. 3: 10.5g of bran, 12.5g of corn flour, 25.0g of soybean cake powder, 1.0g of calcium carbonate and MgSO4·7H2O2.0g, sodium chloride 4.4g and water 1.0L.
Medium No. 4: 3.50g of glucose, 0.80g of peptone, 0.50g of yeast powder, 0.35g of monopotassium phosphate and CaCO30.25g and 1.00L of water.
Medium No. 5: 46.0g of corn flour, 35.0g of soybean cake powder, 1.0g of glucose, 3.2g of dipotassium hydrogen phosphate and MgSO4·7H2O0.1g and water 1.0L.
Medium No. 6: corn flour 3.0g, glucose 3.0g, soybean cake powder 3.0g, peptone 10.0g, MgSO4·7H2O0.4g, ferrous sulfate 0.02g and water 1.0L.
Medium No. 7: 3.0g of corn flour, 3.0g of soybean cake powder, 0.5g of glucose, 2.0g of manganese sulfate, 3.0g of dipotassium hydrogen phosphate, 1.5g of monopotassium phosphate and 1.0L of water.
Medium No. 8: 10.0g of glucose, 15.0g of peptone, 15.0g of yeast powder, 1.0g of monopotassium phosphate and MgSO4·7H2O1.5g, sodium chloride 5.0g, water 1.0L.
Medium No. 9: corn flour 10.0g, soybean cake powder 30.0g, NaCl 2.0g, CaCO3 7.0g、k2HPO43.0g and 1.0L of water.
Medium No. 10: 10.0g of peptone, 5.0g of beef extract, 10.0g of sucrose and Na2HPO4 4.0g、MgSO40.2g and 1.0L of water.
Medium No. 11: 30.0g of bran, 1.5g of ammonium sulfate, 5.0g of sodium chloride, 1.0g of calcium chloride and 1.0L of water.
Medium No. 12: 20.0g of bran, 20.0g of peptone, 1.0g of monopotassium phosphate, 0.5g of ammonium sulfate and 1.0L of water.
Medium No. 13: 48.0g of corn flour, 4.0g of corn steep liquor, 8.0g of ammonium sulfate, 10.0g of calcium carbonate and 1.0L of water.
Medium No. 14: 10.0g of soluble starch, 20.0g of corn flour and KH2PO4 1.0g、(NH4)2SO40.5g, peptone 20.0g, and water 1.0L.
Medium No. 15: 10.0g of soluble starch, 10.0g of glucose, 20.0g of peptone and KH2PO4 1.0g、(NH4)2SO40.5g and 1.0L of water.
Medium No. 16: 10.0g of soluble starch, 5.0g of glucose, 20.0g of peptone and KH2PO4 1.0g、(NH4)2SO40.5g and 1.0L of water.
Medium No. 17: soybean cake powder 20.0g, peptone 20.0g, KH2PO4 1.0g、(NH4)2SO40.5g and 1.0L of water.
Medium No. 18: glucose 10.0g, peptone 10.0g, KH2PO4 0.2g、MgSO40.2g and 1.0L of water.
Medium No. 19: 42.0g of bran, 40.0g of cane sugar, 10.0g of NaCl and 1.0L of water.
20.0g of No. 20 culture medium bran, 10.0g of peptone, 5.0g of glucose and KH2PO4 1.0g、MgSO40.5g and 1.0L of water.
Medium No. 21: 20.0g of bran, 15.0g of peptone, 5.0g of glucose and KH2PO4 1.0g、MgSO40.5g and 1.0L of water.
Medium No. 22: 12.50g of soluble starch, 12.50g of peptone and KH2PO4 0.25g、MgSO40.50g and 1.00L of water.
Medium No. 23: 15.0g of soluble starch, 15.0g of soybean cake powder, 15.0g of NaCl and 1.0L of water.
Medium No. 24: corn flour 13.0g, peptone 15.0g, CaCO3 0.8g、NaH2SO4 2.0g、Na2HSO44.0g and 1.0L of water.
Medium No. 25: 80.0g of soybean cake powder, 30.0g of corn flour and KH2PO4 1.0g、MgSO4 0.5g、FeSO40.15g、MnSO40.05g and 1.0L of water.
Medium No. 26: soybean cake powder 13.0g, NaCl 12.0g, MnSO40.25g and 1.0L of water.
Medium No. 27: 15.0g of sucrose, 2.5g of yeast powder, 2.5g of peptone, 5.0g of NaCl and 1.0L of water.
Medium No. 28: glucose 30.0g, yeast powder 15.0g, Na2HPO4 2.0g、NaH2PO4 1.0g、MgSO41.0g and 1.0L of water.
Medium No. 29: glucose 10.0g, yeast powder 5.0g, MgSO41.0g and 1.0L of water.
Medium No. 30: 12.5g of soluble starch, 12.5g of peptone and KH2PO4 0.25g、MgSO40.5g, 10.0g of glucose and 1.0L of water.
Medium No. 31: 12.5g of soluble starch, 12.5g of peptone and KH2PO4 0.25g、MgSO40.5g, 5.0g glucose and 1.0L water.
Medium No. 32: 15.0g of soluble starch, 15.0g of soybean cake powder, 15.0g of NaCl, 10.0g of glucose and 1.0L of water.
Medium No. 33: 15.0g of soluble starch, 15.0g of soybean cake powder, 15.0g of NaCl, 5.0g of glucose and 1.0L of water.
As can be seen from Table 8 and FIG. 6, HG-8 strain had the highest number of cells in medium No. 22, which was 2.7X 109CFU/mL. Therefore, the basal fermentation medium with HG-8 strain as No. 22 medium was selected, and the medium components were soluble starch 12.5g, peptone 12.5g, and K2HPO4 0.25g、MgSO40.5g and 1L of water.
Table 8: preliminary screening of the culture medium
(3) Fermentation medium composition optimization orthogonal assay
After screening the basic culture medium, L is adopted9(34) An orthogonal design test is carried out on the formulation No. 22 with better fermentation property, 4 orthogonal tests with 3-factor levels are set, 9 tests are carried out totally, 3 times of repetition is set for each test, and the level of each factor of the orthogonal tests is set as shown in a table 9.
Table 9: factor and horizontal design of HG-8 strain fermentation medium formula orthogonal test
The results of the orthogonal test of the HG-8 strain fermentation medium are shown in Table 10, and the results show that the fermentation character of the formula No. 6 is the best: soluble starch 12.5g, peptone 9.50g, K2HPO4 0.40g，MgSO40.5g and 1.00L of water. The number of the bacteria in the fermentation liquor reaches 210 multiplied by 108cfu/mL, which was statistically analyzed by SPSS, was significantly different at the 0.01 level.
Table 10: results of orthogonal experiments on HG-8 strain fermentation Medium
SPSS analysis is carried out on all factors, and the soluble starch reaches a remarkable level; the influence of other factors on the number of the bacteria in the fermentation liquor is not at a significant level (Table 11). And (3) integrating the influence results of various factors and levels thereof on the number of the strains to obtain the optimal fermentation medium formula (g/L): soluble starch 12.5g, peptone 9.50g, K2HPO4 0.40g，MgSO40.5g and 1.00L of water.
Table 11: analysis of influence of various factors in fermentation medium on thallus number of HG-8 strain fermentation liquor
Example 9: preparation of HG-8 microbial inoculum
(1) Activating strains: streaking the Bacillus pumilus HG-8 stored at low temperature on an LB solid culture medium plate, and culturing at 30 ℃ for 24 h; and selecting a single colony of the HG-8 strain, streaking again, transferring to an LB solid culture medium plate, and culturing at 30 ℃ for 24 hours for later use.
Solid LB medium formula: 10.0g of peptone, 5.0g of yeast powder, 10.0g of NaCl, 15.0g of agar and 1.0L of water.
(2) Preparing a first-level seed solution: scraping 2 rings of the activated bacterial lawn of the HG-8 strain obtained in the step (1) by using an inoculating ring, inoculating the bacterial lawn in an LB liquid culture medium, culturing at 30 ℃ at 200r/min for 12h by using a shaking bottle to obtain a first-stage seed solution.
LB liquid medium formula: 10.0g of peptone, 5.0g of yeast powder, 10.0g of NaCl and 1.0L of water.
(3) Preparing a secondary seed liquid: inoculating the primary seed liquid prepared in the step (2) into a seed tank according to the proportion of 1.5 percent (volume percentage) of the secondary seed culture medium for fermentation culture; the rotation speed is 120r/min, the temperature is 30 ℃, the tank pressure is 0.05-0.06 MPa, and the culture is carried out for 12 hours to be used as secondary seed liquid.
The formula of the secondary seed culture medium is as follows: soluble starch 12.5g, peptone 9.50g, K2HPO4 0.40g，MgSO40.5g and 1.00L of water.
(4) Fermentation culture: inoculating the secondary seed liquid obtained in the step (3) into a fermentation culture medium according to the volume ratio of 10% for fermentation culture; the rotating speed is 120r/min, the temperature is 30 ℃, the tank pressure is 0.05-0.06 MPa, and the culture time is 30h, so that the HG-8 strain liquid microbial inoculum is obtained.
The formula of the fermentation medium is as follows: soluble starch 12.5g, peptone 9.50g, K2HPO4 0.40g，MgSO40.5g and 1.00L of water.
(5) Preparing a solid microbial inoculum: and (3) centrifuging the HG-8 strain liquid microbial inoculum obtained in the step (4) at 6000-8000 r/min for 20-30 min, collecting precipitates, pumping the precipitates into a stirrer, slowly adding diatomite or corn flour serving as a carrier matrix into the precipitates, adding 6-8% of the diatomite or corn flour based on the weight of the liquid microbial inoculum, uniformly stirring, performing spray drying, dispersing by an atomizer (about 65 ℃) and quickly evaporating to dryness by high-temperature air (180 ℃), and collecting solid matters in feed liquid by a cyclone separator to obtain the HG-8 strain solid microbial inoculum.
Example 10: pot experiment
And (3) investigating the influence of the HG-8 microbial inoculum on the pH value, the quick-acting potassium content and the growth and development of the rhizosphere soil of the wheat.
The test uses wheat as material, wheat seeds are washed for 2 times by clear water, then soaked for 10 hours by clear water, the wheat seeds with uniform size and shape are sown in plastic pots with the inner diameter of 20cm, 10 seeds are planted in each pot, 40 pots are planted in total, and the test selects acid soil with the pH value of 5.35. And when the wheat seedlings grow to 10cm, 10 wheat seedlings with consistent growth vigor are reserved in each pot, 10mL of HG-8 liquid microbial inoculum is applied to each pot of the treatment group, and a control for applying the same amount of inactivated HG-8 liquid microbial inoculum is set. And when the wheat seedlings grow for 60 days, taking out the whole pots of the wheat seedlings of the treatment group and the control group, respectively and randomly selecting 20 wheat seedlings, gently shaking off soil samples attached to the rhizosphere of the wheat, collecting the soil samples, and measuring the pH value and the quick-acting potassium content of the rhizosphere soil of the wheat. The rhizosphere of the wheat is washed clean by tap water, and the growth indexes such as dry, fresh and heavy wheat are measured after the wheat is dried.
Influence of HG-8 microbial inoculum on the pH value of the rhizosphere of the soil. Taking a proper amount of air-dried sample, removing impurities except soil, enabling the sample to completely pass through a test sieve with a pore diameter of 16 meshes, weighing 7g +/-0.1 g of sieved soil sample, placing the sieved soil sample in a 15mL centrifuge tube, and mixing the weighed soil sample with the water in a ratio of 2.5: adding 7mL of carbon dioxide-free water into the water-soil ratio of 1 (injecting a proper amount of deionized water into a conical flask, boiling for 10min, sealing and cooling for later use). After the container was sealed, it was shaken for 5min and then allowed to stand for 1 h. The pH of each soil sample was measured with a pH meter.
Through test determination, the pH value of the wheat rhizosphere soil of the treatment group applied with the HG-8 microbial inoculum is 6.32, and the pH value of the soil of the control group is 5.96. Test results show that the pH value of the rhizosphere soil of the wheat can be obviously improved by applying the HG-8 microbial inoculum. The test results are shown in Table 12.
Influence of HG-8 microbial inoculum on the content of quick-acting potassium in soil. Weighing 5g of air-dried soil with sieve pores smaller than 1mm by using a hundredth balance, putting the soil into a 150mL plastic leaching bottle, and adding 50mL of 1mol/L neutral NH4Shaking OAc solution with cover on oscillator for 30min, taking out and filtering, placing filtrate in a small triangular flask, measuring with potassium standard series on flame photometer, recording galvanometer reading, and measuring with standard curveAnd finding out the corresponding concentration on line, and calculating the content of the soil quick-acting potassium.
And (4) calculating a result: the soil quick-acting potassium content is calculated according to the following formula by checking the value (Kmg/L) from the standard curve.
Soil quick-acting potassium
In the formula: Kmg/L-values found on the standard curve;
v-volume of added leach liquor, m 1;
m-mass of sample, g.
And (3) test results: the content of the quick-acting potassium in the soil of the treatment group applied with the HG-8 microbial inoculum is 82.63mg/kg, which is obviously higher than that of a control group (65.93mg/kg), and is increased by 25.33 percent compared with the control group; the soil pH of the treated group was 6.32 significantly higher than the control group (5.96). Test results show that the pH value of the soil and the content of quick-acting potassium can be obviously improved by applying the HG-8 microbial inoculum. The test results are shown in Table 12.
HG-8 influence of the microbial inoculum on the growth and development of wheat. Test results show that the fresh weight and the dry weight of the wheat seedlings in the treatment group are significantly different from those in the control group, and the fresh weight and the dry weight of the wheat in the treatment group applied with the HG-8 microbial inoculum are increased by 32.41 percent and 35.65 percent respectively compared with those in the control group. The results are shown in Table 12.
Table 12: influence of HG-8 microbial inoculum on pH value of wheat rhizosphere soil, quick-acting potassium and seedling growth
Note: the data in the table are mean ± sd of 3 replicates. The data suffixes in the same row differ alphabetically to indicate significant differences (p <0.05) between different treatment groups, and the letters are the same to indicate insignificant differences between different treatment groups.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> bacillus pumilus with functions of improving acid soil and removing potassium and preparation and application of microbial inoculum thereof
<130> 2019
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1443
<212> DNA
<213> 16S rDNA sequence of HG-8 Strain
<400> 1
ctttggtgct ataatgcagt cgagcggaca gatgggagct tgctccctga tgttagcggc 60
ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc cgggaaaccg 120
gggctaatac cggatggttg tctgaaccgc atggttcaga cataaaaggt ggcttcggct 180
accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg ctcaccaagg 240
cgacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag acacggccca 300
gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc tgacggagca 360
acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg gaagaacaag 420
tgccgttcaa atagggcggc accttgacgg tacctaacca gaaagccacg gctaactacg 480
tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag 540
ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg gggagggtca 600
ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg tagcggtgaa 660
atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct gtaactgacg 720
ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa 780
acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta acgcattaag 840
cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac gggggcccgc 900
acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga 960
catcctctga caatcctaga gataggacgt ccccttcggg ggcagagtga caggtggtgc 1020
atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080
ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt gacaaaccgg 1140
aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct 1200
acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca caaatctgtt 1260
ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg 1320
gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 1380
agagtttgta acacccgaag tcggtgaggt aaccttttag gagccagccg ccgaaggtga 1440
cct 1443
<210> 2
<211> 893
<212> DNA
<213> gyrB sequence of HG-8 Strain
<400> 2
gacgcgtgct tttgagtgct ttctctgttg gaatcgggtg acggtattga tggatccggt 60
catccgttgc cacaagcagg acttctatcg tcatcccttg tgcaatggct ctttgagcaa 120
taagcgaagc aatttcgtgt tcggtctgcc ctggcacgat ttcttgacag acactttcta 180
caatgaccgc tgtttcatga caaaggattt catattgact gagcagcgag gcagtaagga 240
tcgatcgaac atgtgacaaa tccggctcga tccattcgaa tccatccatt tgaatgtccg 300
ccccaattcg tttacctttt gtgagttctt gaatgtaggg ggtctcatcc tcataccaat 360
ccattgtgac cacctgaaag ggatgatcga catacgccat ttcctcttca atgatccgct 420
tttcttccat ttgattcaca atgaggacga ggtcgtcttt taagacgagc agctggcaca 480
cgccctctgg cgtatttaac acaatgtgat tccttcttcc gcctgtcagc caggagaagt 540
tatttcgctt ttgaatcaag acaccatcaa gctgcttttc ttccattagc tctcttactt 600
gttcaatcat ctaatcacct gcctgtatga agtgtttgaa tgaaacggtt aaaagctgtt 660
ttcccttgct cagttctttt aaacacaccc gcctgcgaga gaattcgaga agaaatagag 720
ccaatttctt gttgtaaaat ggtttcggta ttgtcctctg taaaggtgtt ctcttcaagc 780
agtcgctttg cccatgtttc gtgtttcatg atagcaggac gggctttgat ttcttcaaga 840
ggactttcgg atagcaatgc ctcttttaat tggcttaact cctctgctat ctg 893
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
ctacggcaag gcgacgctga cg 22
Claims (9)
1. Bacillus pumilus (A. pumilus)Bacillus pumilus) HG-8, having a biological accession number: CGMCC NO. 18620.
2. Comprises the Bacillus pumilus of claim 1, (B), (C)Bacillus pumilus) HG-8 microbial inoculum.
3. The microbial inoculum according to claim 2, which further comprises an auxiliary material and/or a carrier.
4. The microbial inoculum according to claim 2 or 3, wherein the formulation of the microbial inoculum is liquid, granule, powder, wettable powder or water dispersant.
5. Bacillus pumilus (B.) (B.pumilus)Bacillus pumilus) The preparation method of the HG-8 microbial inoculum is characterized by comprising the following steps:
the Bacillus pumilus of claim 1, (A), (B), (C), (Bacillus pumilus) Inoculating the HG-8 seed solution into a fermentation culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: culturing at the rotation speed of 120r/min, the temperature of 30-32 ℃ and the pot pressure of 0.05-0.06 MPa for 20-30 h to obtain the bacillus pumilus ((B.), (Bacillus pumilus) HG-8 fermentation liquor;
the fermentation medium comprises the following components: soluble starch 12.5g, peptone 9.50g, K2HPO4 0.40g，MgSO40.5g and 1.00L of water.
6. The method of claim 5, wherein the Bacillus pumilus (B.), (B.)Bacillus pumilus) The inoculation amount of the HG-8 seed solution is 8-12% by volume.
7. The method according to claim 5, wherein the Bacillus pumilus strain is Bacillus pumilus (Bacillus pumilus), (Bacillus pumilus, Bacillus subtilis, or Bacillus subtilis)Bacillus pumilus) The preparation of the seed liquid of HG-8 comprises the following steps: strain activation, first-stage seed liquid preparation and second-stage seed liquid preparation.
8. The method of claim 7, wherein the bacteria are activated to: bacillus pumilus to be preserved at low temperature (Bacillus pumilus) Streaking HG-8 on an LB solid culture medium plate, and culturing for 24-36 h at 28-32 ℃; selecting a single colony, streaking again, transferring to an LB solid culture medium flat plate, and culturing at 28-32 ℃ for 24-36 h for later use;
the first-stage seed liquid is prepared by the following steps: inoculating the activated HG-8 strain lawn in an LB liquid culture medium, performing shake culture at the temperature of 28-32 ℃ for 12-16 h at 200-220 r/min to obtain a first-stage seed solution;
the second-stage seed liquid is prepared by the following steps: inoculating the primary seed liquid into a secondary seed culture medium according to the volume percentage of 1-2% for fermentation culture, wherein the fermentation culture conditions are as follows: culturing at the rotation speed of 120r/min, the temperature of 30-32 ℃ and the tank pressure of 0.05-0.06 MPa for 12-16 h to serve as secondary seed liquid;
the formula of the secondary seed culture medium is as follows: soluble starch 12.5g, peptone 9.50g, K2HPO40.40g，MgSO40.5g and 1.00L of water.
9. The Bacillus pumilus of claim 1, (b)Bacillus pumilus) Use of HG-8 or a microbial agent according to any one of claims 2 to 4) in at least one of the following 1) to 4):
1) the pH value of the acid soil is increased, and the acid soil is improved;
2) promoting the growth of crops under the condition of acid soil;
3) converting the insoluble potassium to soluble potassium;
4) preparing a biological organic fertilizer and/or a composite microbial fertilizer;
the crop is wheat.
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