CN117402789A - Bacillus laterosporus and application thereof in soil improvement - Google Patents
Bacillus laterosporus and application thereof in soil improvement Download PDFInfo
- Publication number
- CN117402789A CN117402789A CN202311426595.2A CN202311426595A CN117402789A CN 117402789 A CN117402789 A CN 117402789A CN 202311426595 A CN202311426595 A CN 202311426595A CN 117402789 A CN117402789 A CN 117402789A
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- bacillus
- soil
- bacillus laterosporus
- llh
- fermentation
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Classifications
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
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- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Abstract
The invention relates to the technical field of functional microorganism screening and application, and in particular provides bacillus laterosporusBacillus spporidia) And its application in soil improvement. The bacillus laterosporus is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan, china at the year of 2022, 11 and 14: m20221788 has nitrogen fixation and phosphorus dissolution functions, and can effectively improve soil fertility, improve soil structure, promote crop growth and increase yieldThe quality and quantity can be widely applied to the agricultural field.
Description
Technical Field
The invention relates to the technical field of natural microorganism screening and application, and particularly provides bacillus laterosporus and application thereof in soil improvement.
Background
In recent years, as ecological environmental awareness increases and sustainable development strategies are continuously implemented worldwide, microbial agents are becoming a research area of great concern. The microbial agent has remarkable soil fertility improvement effect, and a large number of researches show that the microbial agent promotes crop growth and improves yield.
The microbial inoculum has the following main effects on soil and crops: (1) increasing the quality and yield of crops. The microbial agent has remarkable synergism on improving the quality of crops, and the active microorganisms contained in the microbial agent can improve the soil environment and activate fungus communities in the soil, so that the absorption capacity of root systems of the crops and the nutrient utilization rate are improved, and the crops produce high-quality fruits. In addition, the microorganism can decompose and synthesize organic matters through the actions of oxidation, nitrification, denitrification and the like, so as to promote the growth of crops. The microbial agent can also improve the quality and taste of crops, such as taste, color, nutritional value and the like. The microbial agent can improve the absorption efficiency of crops on the basis of improving the soil environment and regulating the functions of soil microbial communities, so that the crop yield is obviously improved. (2) enhancing stress resistance of crops. The microbial agent can improve the stress resistance of crops, and the active microorganisms contained in the microbial agent can improve the soil environment, increase the nutrient element absorption capacity of plants and promote the healthy development of plant systems, so that the capability of plants for resisting various pressures and stress is improved. The main ways of increasing crop stress resistance of microbial agents are to improve the water utilization efficiency, improve the crop resistance exposed under the condition of nutrient deficiency, improve the capability of resisting diseases and insect pests, enhance the cold resistance and the like. (3) reducing fertilizer application. The microbial agent can improve soil quality, improve the capability of crops for resisting stress conditions (such as drought or low temperature), reduce the requirement for using chemical fertilizers, and improve a series of problems caused by irregular use of chemical fertilizers for a long time. The active microorganisms contained in the microbial agent can repair the pH value of soil, maintain the humidity of the soil, and increase the mechanical stability and texture of the soil, thereby being beneficial to the reasonable distribution and utilization of nutrient elements such as nutrient level, nitrogen release speed and the like and reducing pollution caused by excessive fertilization. The active microorganisms in the microbial agent can also release various growth factors to stimulate the rapid development of the root system, meanwhile, the micropore and the root system are utilized to cooperate to effectively maintain the microbial diversity of the soil layer, and the adverse competition of toxic substances and dissolved nutrients in the root zone is reduced, so that the use of chemical fertilizers is reduced by improving the absorption efficiency of the root system to nutrient elements. And (4) restoring the soil fertility. The microbial agent can restore soil fertility, and mainly because various active microorganisms contained in the microbial agent can improve the physical structure of soil, increase the decomposition and mineralization speed of organic matters, improve the environmental conditions of the soil and the like through various ways, the soil is more suitable for plant growth, and the soil fertility is positively related to the types of microorganisms. Microbial agents contain a variety of beneficial microorganisms, and a large number of organic matters naturally exist in soil, and the microorganisms can promote the decomposition and mineralization of the organic matters, so that abundant nutrient elements are released for the growth of crops. Meanwhile, a plurality of bacteria with enzyme producing function are found at home and abroad, and the bacteria can utilize heavy metal ions in soil, and important nutrients such as loosening and fixing nitrogen oxide grains Le Suan and the like to realize the full potential of deep circulation excavation of the nutrients buried at the bottom among soaked soil particles. The microbial agent can also secrete various organic matters, cell wall flocculating agents and other chemical substances to construct a complete bottom and pore space. By doing so, the plant can be helped to establish a strong and stable soil environment, so that the development of the root system of the crop is promoted, the root system can absorb nutrients more positively, and the crop can fully utilize the nutrients in the soil. The active microorganisms contained in the microbial agent can reduce the pH value of the soil, improve the weathering degree of the soil and increase the permeability of the soil, thereby improving the structure of the soil and creating good conditions for the growth of plant root systems.
At present, many developments in the research of microbial agents have been made, for example Zhao Xiaoyan and the like, a soil restoration composite microbial agent containing dyford bacteria D39 and Jun sheet bacteria M1031 is invented, and experiments show that the Jun sheet bacteria M1031 has a certain vitamin stability and synergy on the degradation of phoxim by dyford bacteria D39, and in the long-term degradation process, the Jun sheet bacteria M1031 is helpful for maintaining good activity of dyford bacteria D39 and promoting the degradation of phoxim. The soil remediation compound microbial inoculum has the advantages of high degradation efficiency, long activity period and wide application range. Wang Xinge and the like provide a bacillus subtilis XHS0035Kc (with the preservation number of CGMCCNO.9434) suitable for greenhouse soil remediation, and the bacillus subtilis is combined with pseudomonas fluorescens, bacillus mucilaginosus and saccharomycetes lock to prepare a soil remediation composition, so that the soil is effectively improved and activated, harmful substances in the soil are degraded, the salinization problem of the soil is solved, and the current situation that the soil nutrient in a greenhouse of an actual facility is unbalanced, salinized, sandized or hardened is serious is solved.
At present, further excavation of agricultural microbial strain resources capable of effectively improving soil is a research hotspot in the field.
Disclosure of Invention
The invention aims to provide bacillus laterosporusBacillus spporidia) And its application in soil improvement. The bacillus laterosporus has the functions of nitrogen fixation and phosphorus dissolution, can effectively improve soil fertility, improve soil structure, promote crop growth, and improve yield and quality, and can be widely applied to the agricultural field.
One aspect of the invention provides a bacillus laterosporus LLH-C6%Bacillus spporidia LLH-C6) strain, which was deposited at the chinese collection of typical cultures at university of armed chinese in chinese at 11 months 14 of 2022, with a deposit number of cctccc NO: m20221788.
The colony morphology of the bacillus laterosporus LLH-C6 strain is shown in figure 1.
The invention also provides application of the bacillus laterosporus LLH-C6 strain in biological fertilizer production.
The invention also provides application of the bacillus laterosporus LLH-C6 strain in the production of soil conditioners.
The invention also relates to a preparation method of the bacillus laterosporus LLH-C6 strain bacterial powder, which comprises the following steps:
(1) Strain activation and seed liquid preparation
Inoculating the bacillus laterosporus LLH-C6 strain into an LB plate culture medium, performing activation culture for 2d at 37 ℃, transferring into an LB liquid culture medium, and culturing for 24h at 37 ℃ and 180 rpm; transferring into seed culture medium of seed tank, fermenting at 37deg.C and 200rpm for 18 hr to obtain seed solution;
(2) Preparation of fermentation broth
Inoculating the seed solution prepared in the step (1) into a fermentation medium according to the volume ratio of 5%, and stopping fermentation when the spore formation rate is more than 90% under the conditions of 37 ℃ and 180rpm to obtain fermentation liquor;
(3) Spray drying
Centrifuging the fermentation liquor prepared in the step (2) at 5000rpm for 10min, removing fermentation supernatant, and performing spray drying on bacterial mud to obtain bacterial powder.
The seed culture medium in the step (1) comprises the following components in percentage by mass: 2.5% of glucose, 5.5% of corn meal, 3.5% of bean cake meal, 0.3% of potassium dihydrogen phosphate and 0.01% of manganese chloride.
The fermentation medium in the step (2) comprises the following components in percentage by mass: 2.5% of glucose, 6.0% of corn meal, 7.5% of bean cake meal, 0.3% of potassium dihydrogen phosphate and 0.02% of manganese chloride.
The invention also provides a soil conditioner which comprises bacillus laterosporus LLH-C6 strain.
The soil conditioner further comprises any one or more of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus methylotrophicus, bacillus megaterium, bacillus pumilus, lactobacillus plantarum, bacillus mucilaginosus, aspergillus niger, trichoderma viride, trichoderma harzianum, paecilomyces lilacinus, rhodopseudomonas palustris and photosynthetic bacteria.
The live bacterial amount of the bacillus laterosporus LLH-C6 strain in the soil conditioner is not less than 10 9 CFU/g。
The invention also relates to application of the soil conditioner in agricultural production.
The bacillus laterosporus LLH-C6 obtained by screening from the soil has nitrogen fixation capability and obvious phosphorus dissolving effect, is fermented in a Meng Jinna inorganic phosphorus liquid culture medium for 4 days, and has the highest effective phosphorus content of 979.3mg/L and very obvious phosphorus dissolving effect.
Bacillus laterosporus LLH-C6 can produce chitinase and cellulase in high yield, and the enzyme activities of the chitinase and the cellulase in the fermentation supernatant respectively reach 15.4U/mL and 5.4U/mL; can efficiently secrete the indoleacetic acid, the yield of the indoleacetic acid is 25.67 +/-0.43 mg/L in a DF culture medium, and the yield of the indoleacetic acid is 56.32+/-1.31 mg/L in a DF+ culture medium, so that unexpected technical effects are achieved.
Bacillus laterosporus LLH-C6 can effectively improve soil fertility, promote soil to form a granular structure, improve the internal pore space of soil, increase soil permeability, promote crop growth and realize yield and income increase. Compared with a control group, the organic matter content in the soil of a treatment group to which the bacillus laterosporus LLH-C6 bacterial powder is applied is improved by 13.0% -16.4%, the total porosity and capillary porosity of the soil are respectively improved by 33.6% -38.0% and 29.2% -36.3%, the yield of corn is increased by 28.2% -37.3%, the yield of muskmelon is also generally improved by 12.3% -21.8%, and the content of soluble solids and vitamin C in the muskmelon is generally improved by 21.3% -29.3% and 14.0% -38.0%, so that unexpected technical effects are achieved.
In addition, the bacillus laterosporus LLH-C6 has remarkable effect of improving the acidified soil, can keep the soil loose and improves the soil structure. The pH change of the acid soil of the treatment group to which the bacillus laterosporus LLH-C6 is applied before and after planting is most obvious, the pH is changed from acidity to weak acidity, and the pH is increased by 1.0; the soil volume weight is also obviously reduced.
In conclusion, the bacillus laterosporus LLH-C6 provided by the invention can be singly used or combined with beneficial microorganisms such as bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens and the like to be used for producing biological fertilizers or soil conditioners, and can be widely applied to the field of agricultural planting.
Drawings
FIG. 1 is a colony morphology of Bacillus laterosporus LLH-C6.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The formula of the culture medium used in the embodiment of the invention is as follows:
LB solid medium: 10g tryptone, 5g yeast extract powder, 10g sodium chloride, 1000mL distilled water, 20g agar powder and pH value of 7.2-7.4.
LB liquid medium: 10g tryptone, 5g yeast extract powder, 10g sodium chloride, 1000mL distilled water and pH value of 7.2-7.4.
DF medium: 5.00g of peptone, 1.50g of yeast extract, 1.50g of beef extract, 5.00g of NaCl, 1000mL of distilled water and pH 9.0;
df+ medium: 5.00g of peptone, 1.50g of yeast extract, 1.50g,NaCl 5.00g,0.50g/L of tryptophan as beef extract, 1000mL of distilled water and pH of 9.0.
The invention is further illustrated below with reference to examples.
Example 1 isolation and screening of Nitrogen-fixing and phosphorus-dissolving microorganisms in soil
1. Soil sample
In the vegetable planting base of Qingdao city, the soil sample is sampled by five-point method, the sample collecting depth is 5-20 cm, and after the sundries such as grass, wood dust and stone are picked out, the soil sample is stored in a refrigerator at 4 ℃ for standby.
2. Azotobacter primary screening
The culture medium for screening the nitrogen-fixing bacteria is an Ababetes nitrogen-free solid culture medium (Ashby), and the specific preparation method of the culture medium comprises the following steps: 10g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 0.2g of sodium chloride, 5g of calcium carbonate, 0.1g of calcium sulfate dihydrate, 18g of agar, pH of 6.8-7.0 and water supplementing to 1000ml; sterilizing at 121deg.C for 20min.
Weighing 10g of soil sample, placing the soil sample in a 250ml triangular flask filled with a proper amount of glass beads and 90ml of sterile water, oscillating at 30 ℃ and 180rpm for 20min, and standing for 10min to obtain soil suspension; diluting the soil suspension to 10 by adopting a gradient dilution method -6 Then, sucking 0.1ml of diluted soil suspension, uniformly coating on an Ashby solid culture medium, and placing in a 30 ℃ incubator for inversion culture for 24 hours; different types of typical single colonies are picked out, purified for multiple times by a flat plate, and stored on an Ashby solid culture medium inclined plane at 4 ℃ for later use.
The total nitrogen-fixing bacteria 14 strains are obtained by this round of screening and are respectively named as C1, C2, C3, … …, C13 and C14.
3. Phosphate-solubilizing bacteria compound sieve
And (3) inoculating 14 strains of nitrogen-fixing bacteria obtained by primary screening on a Meng Jinna inorganic phosphorus solid culture medium plate respectively under a sterile condition, culturing for 4 days at 30 ℃, and observing whether transparent rings appear around colonies.
3 strains C4, C6 and C10 with the largest transparent circles are selected and respectively inoculated into 50mL Meng Jinna inorganic phosphorus liquid culture medium, the temperature is 30 ℃, the rpm is 200, the culture is carried out for 4 days, and simultaneously Meng Jinna liquid culture medium without any bacteria is used as a control group.
(1) Drawing of phosphorus standard curve
Sequentially sucking 0.0, 0.2, 0.4, 0.8, 1.6, 2.0, 3.2 and 4.0ml of 5mg/l phosphorus standard solution into a test tube, then adding 2ml of molybdenum-antimony anti-color reagent into each test tube, adding distilled water to a volume of 20ml, shaking uniformly, standing for 20min, and measuring absorbance at 700nm wavelength. The phosphorus concentration in each tube at this time was divided into: 0.00, 0.05, 0.10, 0.20, 0.40, 0.50, 0.80, 1.00mg/l. And drawing a phosphorus standard curve by taking the phosphorus concentration as an abscissa and the absorbance as an ordinate.
The effective phosphorus content calculation formula is as follows:
effective phosphorus (%) = (reading of phosphorus in color development solution×volume of color development solution×division multiple) ×100/(w×10) 6 ）。
Wherein: the reading of the phosphorus (P) in the color development liquid is the reading of the phosphorus in the color development liquid obtained by looking up the phosphorus standard curve; color developmentThe liquid volume is 50 mL; the division multiple is fermentation liquor volume (mL)/absorption fermentation liquor (mL); w is the mass (refer to each triangular flask) of organic or inorganic phosphorus compound substances added into the fermentation broth, and g;10 6 In order to convert μg to g.
(2) Determination of available phosphorus content
Respectively taking fermentation culture solutions of C4, C6 and C10 strains, and centrifuging at 4deg.C and 12000 r/min for 15 min; taking 5mL fermentation supernatant into 150 mL conical flask, adding 0.5 mol/L NaHCO 3 45 to mL, adding a scoop of phosphorus-free active carbon, sealing with a sealing film, oscillating for 30 min, and filtering with phosphorus-free filter paper; taking 10 mL filtrate, putting the filtrate into a volumetric flask of 50mL, slowly adding 5mL molybdenum-antimony color-developing resisting agent, uniformly shaking the filtrate at constant volume, standing the filtrate for 30 min, and performing color comparison at 700nm wavelength. And reading out the light absorption value of the liquid to be detected, and substituting the light absorption value into a standard curve and a formula to calculate the content of the effective phosphorus.
The result shows that the content of effective phosphorus in the fermentation supernatant of the C6 strain in the three nitrogen-fixing phosphorus-decomposing bacteria screened by the invention is highest, reaching 979.3mg/L, and the phosphorus-decomposing effect is very obvious.
The C6 strain obtained by screening has nitrogen fixation capacity and obvious phosphorus dissolving effect, is beneficial to improving the soil fertilizer efficiency, and can be widely applied to the field of agricultural planting.
Example 2 identification of the azotometer C6 Strain
2.1 Colony morphology identification
As shown in FIG. 1, the colony of the C6 strain is round, off-white or yellow-white, semitransparent, smooth and neat in edge and provided with a protrusion in the central part; the thallus is thick and short rod-shaped, both ends of the thallus are rounded, and the spore is laterally born, moderately born or nearly moderately born, elliptic and the cyst is enlarged.
2.2 16s rDNA identification
Single colonies of the C6 strain on the plate were picked up and cultured in nutrient broth medium at 37℃for 18 hours, then 500ul of strain fermentation broth was taken and the genome of the strain was extracted using the kit. The genome is used as a template, a specific primer sequence is designed, and a 16s rDNA sequence of the C6 strain is amplified by PCR and sequenced.
16srDNA sequences of the C6 Strain were entered in NCBI databaseBLAST comparison with Bacillus laterosporusBacillus spporidia) Is the highest in similarity. Thus, the C6 strain was preliminarily determined to be Bacillus laterosporus.
2.3 Physiological and biochemical characteristics
Physiological and biochemical characteristic test identification of C6 strain: performing sugar fermentation test; a catalase test; gelatin liquefaction test; ammonia production test; glucose acidogenesis and gas production test; hydrogen sulfide testing; indole test; motility test.
In conclusion, in combination with the colony morphology, physiological and biochemical characteristics and molecular biological identification results of the C6 strain, the applicant can confirm that the screened C6 strain is a bacillus laterosporus strainBacillus spporidia) The bacillus laterosporus strain is named as bacillus laterosporus LLH-C6%Bacillus spporidia LLH-C6）。
The applicant prepared the bacillus laterosporus LLH-C6 at 2022, 11 and 14 daysBacillus spporidia LLH-C6) strain preserved in China center for type culture collection (CCTCC NO) of university of Wuhan, china, with the preservation number of CCTCC NO: m20221788.
EXAMPLE 3 evaluation of the enzyme Producation ability of Bacillus laterosporus LLH-C6
Inoculating bacillus laterosporus LLH-C6 into LB culture medium, and performing activation culture for 24 hours at 37 ℃ to obtain an activated strain; inoculating the activated strain into fermentation medium (glucose 20g, bean cake powder 10g, corn steep liquor 5g, water 1L, pH 7.0. When in use, 0.13Mpa high pressure sterilization for 40min at 121deg.C), culturing for 4d at 35deg.C and 220r/min to obtain fermentation broth; the Bacillus laterosporus LLH-C6 fermentation broth was centrifuged at 12000rpm at 4℃for 10 minutes, and the supernatant was taken.
1. Chitinase enzyme activity assay
The enzyme activity is measured by referring to the method of Dai Dehui and the like, and corresponding reasonable modification is carried out on the basis: taking 1mL treated fermentation supernatant and 1mL of 1% colloidal chitin-phosphate buffer solution, carrying out water bath at 45 ℃ for 1 h, boiling in a boiling water bath for 40min, adding 2mL of 3,5-dinitrosalicylic acid (3, 5-dinitrosalicylic acid, DNS) after terminating the reaction, boiling for 10min, fixing the volume to 10 mL, centrifuging, and measuring the OD value of the supernatant at the wavelength of 540 nm. The product of the enzymatic reaction, chitooligosaccharide, undergoes oxidation-reduction reaction with DNS, develops a reddish brown color in boiling condition, and detects enzyme activity at a wavelength of 540, 540 nm. Under the above conditions, the amount of enzyme used in the catalytic production of 1. Mu.g of glucosamine per minute was defined as 1 enzyme activity unit (U).
The measurement result shows that the chitinase activity in the bacillus laterosporus LLH-C6 fermentation supernatant reaches 15.4U/mL.
2. Cellulase enzyme activity assay
Definition of enzyme activity: the amount of enzyme required to degrade and release 1. Mu. Mol of reducing sugar per minute from a sodium hydroxymethyl cellulose solution with a concentration of 5 mg/ml at 50 ℃ and a pH value of 6.0 is one enzyme activity unit U, and the reducing sugar is glucose equivalent.
The measuring method comprises the following steps: three test tubes were each added with 0.5 mL CMC substrate and preheated in a water bath at 50℃for 5min with the enzyme solution to be tested. And adding 0.5. 0.5 mL to-be-detected liquid into each of the first test tube and the second test tube, timing, and reacting for 15 min in a water bath at 50 ℃. After the reaction was completed, 1.5 mL of DNS reagent was added to each of the three test tubes, and the third test tube was supplemented with 0.5. 0.5 mL of enzyme solution to be tested. After taking out and shaking three test tubes, the reaction was carried out in a boiling water bath for 5 min. Cool rapidly to room temperature and set with water to 5.0. 5.0 mL. The absorbance of the first test tube and the second test tube is preferably 0.25-0.35 under the condition of 540 and nm wavelength by taking the third test tube as a control. The absolute value of the difference between the absorbance of the enzyme liquid reaction liquid to be detected and the absorbance of the enzyme liquid reaction liquid is controlled to be not more than 0.015.
Enzyme activity calculation formula: x= (glucose equivalent/180/15/0.5) ×n.
Wherein: x-enzyme activity unit, IU/g (mL); 180-glucose converted from micrograms to micromoles; 15-the reaction time of the liquid to be detected and the substrate; 0.5-adding the amount of enzyme to be detected in the reaction; n-dilution fold.
The measurement result shows that the enzyme activity of the cellulase in the bacillus laterosporus LLH-C6 fermentation supernatant reaches 5.4U/mL.
In conclusion, the bacillus laterosporus LLH-C6 provided by the invention can produce chitinase and cellulase in high yield, and has unexpected technical effects.
EXAMPLE 4 evaluation of the ability of Bacillus laterosporus LLH-C6 to produce indoleacetic acid
Indoleacetic acid is a plant growth regulator, and can promote the growth and development of plants, improve the yield and quality of the plants, and can also be used for regulating the growth rhythm of the plants, promoting the dormancy and germination of the plants, improving the adaptability and stress resistance of the plants and enhancing the disease resistance and stress resistance of the plants.
The purpose of this example is to test the ability of Bacillus laterosporus LLH-C6 to produce indoleacetic acid.
Inoculating bacillus laterosporus LLH-C6 into LB culture medium, and performing activation culture for 24 hours at 37 ℃; inoculating bacillus laterosporus LLH-C6 bacterial liquid into DF culture medium (pH 9.0) and DF+ culture medium (0.50 g/L tryptophan is added into DF culture medium with pH 9.0) respectively according to the inoculum size of 2%, and culturing for 7 days at 35 ℃ and 150rpm in shake flask; the fermentation broth was centrifuged at 12000rpm at 4℃for 5min, 30mL of supernatant was taken, extracted with twice the volume of ethyl acetate in a constant temperature shaker for 3 times, the extracts were combined, distilled under reduced pressure, then dissolved with 5mL of methanol, fixed in volume, and filtered through a 0.22 μm filter membrane.
Detection instrument: waters2998 high performance liquid chromatography; chromatographic column: agilertZorbaxSB-C18250mm 4.6mm,5 μm; mobile phase, methanol: acetonitrile: 0.6% glacial acetic acid in water (50:5:45, v/v/v); sample injection amount: 20. Mu.L; the flow rate is 0.8mL/min; column temperature: room temperature; detection wavelength: 255nm.
The detection result shows that: in DF medium, the yield of indoleacetic acid in bacillus laterosporus LLH-C6 fermentation broth is 25.67 +/-0.43 mg/L, and in DF+ medium, the yield of indoleacetic acid is 56.32+/-1.31 mg/L.
The results show that the bacillus laterosporus LLH-C6 provided by the invention can efficiently secrete indoleacetic acid, is expected to be applied to the field of biological fertilizers, and improves the yield and quality of crops.
EXAMPLE 5 preparation method of Bacillus laterosporus LLH-C6 powder
(1) Strain activation and seed liquid preparation
Inoculating the bacillus laterosporus LLH-C6 strain into an LB plate culture medium, performing activation culture for 2d at 37 ℃, transferring into an LB liquid culture medium, and culturing for 24h at 37 ℃ and 180 rpm; transferring into seed culture medium (the mass percentage of each component in the culture medium is glucose 2.5%, corn meal 5.5%, bean cake powder 3.5%, potassium dihydrogen phosphate 0.3%, manganese chloride 0.01%, pH 7.0;) of a seed tank, fermenting at 37deg.C and 200rpm for 18 hr to obtain seed solution;
(2) Preparation of fermentation broth
Inoculating the seed solution prepared in the step (1) into a fermentation medium (the mass percentage of each component in the medium is 2.5% of glucose, 6.0% of corn meal, 7.5% of bean cake meal, 0.3% of monopotassium phosphate, 0.02% of manganese chloride and pH 7.0) according to the volume ratio of 5%, and fermenting at 37 ℃ and 180rpm until the spore formation rate is above 90%, and stopping fermenting to obtain a fermentation solution;
(3) Spray drying
Centrifuging the fermentation liquor prepared in the step (2) at 5000rpm for 10min, removing fermentation supernatant, and performing spray drying on bacterial mud to obtain bacterial powder.
Through detection, the live bacterial amount of the bacillus laterosporus LLH-C6 in the prepared bacterial powder is about 3.5X10 10 CFU/g of bacterial powder.
Example 6 Effect of Bacillus laterosporus LLH-C6 on corn yield increase and soil structural improvement
The corn planting test site is selected from red flag village corn planting demonstration sites in Qingdao city of Shandong, and the red flag village is in Qingdao city, and the red flag village is in tidal sand soil, texture sand soil and fertility; maize variety: jade 335; planting test time: 2022, 5.1.day to 2022, 10.1.day.
A 10m×50m area was selected as one experimental area, 15 experimental areas were set in total, and a 1 meter interval was maintained between each experimental area. 5 experimental sections were randomly selected for each treatment group for the experiment.
Conventional soil preparation, shallow tillage and unified machine sowing are carried out before sowing. The base fertilizer is applied with a compound fertilizer with the mass ratio of nitrogen, phosphorus and potassium of 15-5-5, and the application amount is 70 kg/mu.
The following treatment is carried out when corn topdressing is carried out twice on 15 days of 6 months and 20 days of 7 months:
(1) Blank control group: pouring clear water into the roots of wheat;
(2) LLH-C6 bacterial powder treatment group: the bacillus laterosporus LLH-C6 bacterial powder is poured on the root of corn along with water. Wherein:
treatment group 1: the consumption of the bacterial powder is 5.0 kg/mu;
treatment group 2: the consumption of the bacterial powder is 8.0 kg/mu.
When corn is harvested, harvesting, threshing and airing are uniformly carried out, the corn yield in each experimental area is counted, the average corn yield is calculated, the content of organic matters in soil is measured by adopting a potassium dichromate capacity method, the total porosity of the soil and the porosity of a capillary of the soil are calculated by utilizing a ring cutting method, and the results are shown in tables 1 and 2.
TABLE 1 influence of Bacillus laterosporus LLH-C6 on corn yield
| Experimental grouping | Topdressing fertilizer | Average mu yield (kg) |
| Control group | - | 630.5 |
| LLH-C6 bacterial powder treatment group 1 | 5.0 kg/mu | 808.2 |
| LLH-C6 bacterial powder treatment group 2 | 8.0 kg/mu | 865.5 |
From the experimental data in Table 1, compared with the control group, the yield of corn of the two treatment groups to which the bacillus laterosporus LLH-C6 bacterial powder is applied is generally improved, the yield per mu exceeds 800 kg, and the amplification reaches 28.2% -37.3%. Therefore, the bacillus laterosporus LLH-C6 screened by the method can obviously improve the fertilizer efficiency of soil, promote the growth and development of corn, and achieve unexpected technical effects.
TABLE 2 influence of Bacillus laterosporus LLH-C6 on soil structure
| Experimental grouping | Organic matter content | Total porosity of soil | Porosity of soil capillary |
| Control group | 1.46% | 25.32% | 13.25% |
| LLH-C6 bacterial powder treatment group 1 | 1.65% | 33.82% | 17.12% |
| LLH-C6 bacterial powder treatment group 2 | 1.70% | 34.93% | 18.06% |
As can be seen from the experimental data in Table 2, compared with the control group, the organic matter content in the soil of the treatment group to which the bacillus laterosporus LLH-C6 bacterial powder is applied is improved by 13.0% -16.4%, the total porosity of the soil and the capillary porosity are respectively improved by 33.6% -38.0% and 29.2% -36.3%, and the effect is remarkable. Therefore, the bacillus laterosporus LLH-C6 can effectively improve soil fertility, promote soil to form a granular structure, improve the internal pore space of the soil, increase the permeability of the soil, create good conditions for crop growth, finally realize yield and income increase, and achieve unexpected technical effects.
EXAMPLE 7 Effect of Bacillus laterosporus LLH-C6 on melon yield and quality improvement
The experimental place is selected from the vegetable planting base of the Qingdao city, the Ping Jia Ji and the melon planting greenhouse.
The experiment is provided with 9 experiment areas, each experiment area randomly selects 10m multiplied by 10m, and 10 ridges of melons are planted in each experiment area, namely about 500+/-15 strains. And a protection row is arranged between the experimental areas. Experiments were performed in three groups of 3 experimental areas randomly selected for each group.
(1) Blank control group: pouring clear water into the root of melon seedling;
(2) LLH-C6 treatment group: the bacillus laterosporus LLH-C6 bacterial powder prepared in the example 5 is applied with water after melon seedlings are transplanted according to the dosage of 2-4 kg/mu, and is used once every 10 days and three times continuously. Wherein:
LLH-C6 treatment group 1: the consumption of the bacterial powder is 2 kg/mu;
LLH-C6 treatment group 2: the consumption of the bacterial powder is 4 kg/mu.
Picking melons with maturity exceeding 8 in fixed time every morning after the melons enter the maturity period, weighing, and recording the yield of the melons in each experimental area; simultaneously, 15 melons are randomly picked from melons picked in each experimental area every day, and the mass percent content of soluble solids and the content of vitamin C in the melons are detected respectively. After harvesting of all melons, the total yield of melons in each experimental area was counted, and the average total yield of melons in the control group and the LLH-C6 treatment group and the average content of soluble solids and vitamin C in the melons were calculated, respectively, and the specific results are shown in Table 3.
TABLE 3 influence of Bacillus laterosporus LLH-C6 on melon yield and quality
| Experimental grouping | Average yield (kg/mu) | Soluble solids content | VC content mg/100g |
| Blank control group | 2201 | 7.5% | 5.0 |
| LLH-C6 treatment group 1 | 2471 | 9.1% | 5.7 |
| LLH-C6 treatment group 2 | 2683 | 9.7% | 6.9 |
As can be seen from the results in Table 3, the yield of melons in the treated group to which Bacillus laterosporus LLH-C6 bacteria powder was applied was generally increased by 12.3% to 21.8%, the content of soluble solids in melons was generally increased by 21.3% to 29.3%, and the content of vitamin C was increased by 14.0% to 38.0%, as compared with the control group. Therefore, the bacillus laterosporus LLH-C6 provided by the invention can obviously increase the yield of melons, improve the quality and the nutritional value of the melons, and achieve unexpected technical effects.
EXAMPLE 8 improvement effect of Bacillus laterosporus LLH-C6 on acidified soil
Soil acidification seriously affects the normal growth of crops. The acidified soil is hardened, the air permeability is poor, the fertilizer supply capacity is poor, the root system of the planted crops is undeveloped, and the yield is low; long-term and excessive absorption of plants can poison and even die; acidification accelerates loss of mineral nutrient elements in soil, changes soil structure, leads to soil barren, and influences normal development of plants; can also induce plant diseases and insect pests, and reduce the yield of crops.
The aim of the example is to test the improvement effect of bacillus laterosporus LLH-C6 on acidified soil.
Firstly, exposing acid soil with pH of 5.6 for 2 days, sterilizing, and then filling the acid soil into pots, wherein each pot is filled with about 5kg of soil; each treatment was set up with 3 parallel groups of 30 pots each. Transplanting 3 2-3 leaves of corn seedlings in each pot, and managing normal moisture during the test period; the fertilization is carried out for three times on the transplanting day, the 30 th day and the 60 th day respectively, and the specific steps are as follows:
(1) Blank control group: equivalent amount of clean water;
(2) LLH-C6 treatment group 1: inoculating Bacillus laterosporus LLH-C6 bacterial suspension (10) 8 CFU/mL）。
(3) LLH-C6 treatment group 2: inoculating Bacillus laterosporus LLH-C6 bacterial suspension (10) at 100 ml/basin 8 CFU/mL）。
After harvesting corns on day 90, respectively measuring the pH value of each pot of soil of each treatment group, and taking an average value; and the soil volume weight is measured by adopting a ring cutter soil sampling, drying and weighing method, three layers of sampling are divided into 0-10, 10-20 and 20-30 cm, 5 samples are sampled in each layer, and then the average value is calculated. The experimental results are shown in tables 4 and 5
TABLE 4 influence of Bacillus laterosporus LLH-C6 on the pH of acidified soil
| Grouping | pH before planting | Post-planting pH | pH difference |
| Blank control group | 5.6 | 5.5 | -0.1 |
| LLH-C6 treatment group 1 | 5.6 | 6.3 | 0.7 |
| LLH-C6 treatment group 2 | 5.6 | 6.6 | 1.0 |
TABLE 5 Bacillus laterosporus LLH-C6 specific gravity (g/cm) of acidified soil 3 ) Influence of (2)
| Grouping | 0-10cm | 10-20cm | 20-30cm |
| Blank control group | 1.36 | 1.40 | 1.38 |
| LLH-C6 treatment group 1 | 1.30 | 1.33 | 1.31 |
| LLH-C6 treatment group 2 | 1.31 | 1.33 | 1.32 |
The experimental results of tables 4 and 5 show that: the pH change of the acid soil of the treatment group to which the bacillus laterosporus LLH-C6 is applied before and after planting is most obvious, the pH is changed from acidity to weak acidity, and the pH is increased by 1.0; the soil volume weight is also obviously smaller than that of the control group. Therefore, the bacillus laterosporus LLH-C6 provided by the invention has remarkable effect of improving the acidified soil, can maintain the loose soil, improves the soil structure and is beneficial to promoting the growth of crops.
In conclusion, the bacillus laterosporus LLH-C6 provided by the invention can be singly used or combined with beneficial microorganisms such as bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens and the like to be used for producing biological fertilizers or soil conditioners, and can be widely applied to the field of agriculture.
Claims (10)
1. The bacillus laterosporus is characterized in that the collection number of the bacillus laterosporus is CCTCC NO: m20221788.
2. The use of bacillus laterosporus according to claim 1 in the production of biofertilizer.
3. Use of bacillus laterosporus according to claim 1 for the production of soil conditioners.
4. The method for preparing bacillus laterosporus powder according to claim 1, wherein the method comprises the following steps:
(1) Strain activation and seed liquid preparation
Inoculating bacillus laterosporus into LB plate medium, activating and culturing for 2d at 37 ℃, transferring into LB liquid medium, culturing for 24h at 37 ℃ and 180 rpm; transferring into seed culture medium of seed tank, fermenting at 37deg.C and 200rpm for 18 hr to obtain seed solution;
(2) Preparation of fermentation broth
Inoculating the seed solution prepared in the step (1) into a fermentation medium according to the volume ratio of 5%, and stopping fermentation when the spore formation rate is more than 90% under the conditions of 37 ℃ and 180rpm to obtain fermentation liquor;
(3) Spray drying
Centrifuging the fermentation liquor prepared in the step (2) at 5000rpm for 10min, removing fermentation supernatant, and performing spray drying on bacterial mud to obtain bacterial powder.
5. The method of claim 4, wherein the seed medium in step (1) comprises the following components in percentage by mass: 2.5% of glucose, 5.5% of corn meal, 3.5% of bean cake meal, 0.3% of potassium dihydrogen phosphate and 0.01% of manganese chloride.
6. The method according to claim 4 or 5, wherein the fermentation medium in the step (2) comprises the following components in percentage by mass: 2.5% of glucose, 6.0% of corn meal, 7.5% of bean cake meal, 0.3% of potassium dihydrogen phosphate and 0.02% of manganese chloride.
7. A soil conditioner comprising the bacillus laterosporus of claim 1.
8. The soil conditioner of claim 7, further comprising any one or more of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus methylotrophicus, bacillus megaterium, bacillus pumilus, lactobacillus plantarum, bacillus mucilaginosus, aspergillus niger, trichoderma viride, trichoderma harzianum, paecilomyces lilacinus, rhodopseudomonas palustris, photosynthetic bacteria.
9. The soil conditioner according to claim 8, wherein the viable bacterial count of bacillus laterosporus in the soil conditioner is not less than 10 9 CFU/g。
10. Use of a soil conditioner according to any one of claims 7 to 9 in agricultural production.
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