CN117586907A - Saline-alkali tolerant bacillus caldus Bachu85 and application thereof - Google Patents
Saline-alkali tolerant bacillus caldus Bachu85 and application thereof Download PDFInfo
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- CN117586907A CN117586907A CN202311275120.8A CN202311275120A CN117586907A CN 117586907 A CN117586907 A CN 117586907A CN 202311275120 A CN202311275120 A CN 202311275120A CN 117586907 A CN117586907 A CN 117586907A
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- bachu85
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- alkali
- saline
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Classifications
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- 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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
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- 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
- 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
- C09K17/40—Soil-conditioning materials or soil-stabilising materials containing mixtures of inorganic and organic compounds
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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
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Abstract
The invention discloses a salt-tolerant bacillus camptotheciumHalobacillusBachu85 and uses thereof. The strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.2695 at 28/20234. The strain Bachu85 has the capability of salt and alkali resistance, can produce a large amount of auxin IAA (IAA yield reaches 75.65 mu g/mL), and has the functions of fixing nitrogen, decomposing organic phosphorus, producing ACC deaminase, producing catalase, producing siderophore and promoting plant growth. The strain obtained by the invention has important application value in the aspects of relieving the damage of saline-alkali stress to plants, improving the production capacity of the plants, improving the saline-alkali soil, researching and developing microbial fertilizers and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms; in particular to salt-tolerant bacillus caldusHalobacillussp, bachu85 and uses thereof.
Background
Soil salinization is a serious problem, greatly affecting plant growth and yield, and has become an important factor restricting modern agriculture and economic development. Soil salinization refers to the increase of the concentration of water-soluble salts in soil, and is mainly caused by the flooding and infiltration of seawater, low rainfall or high evaporation capacity, etc., and by reducing the soil osmotic potential, plants are difficult to absorb water, and the toxic action of salt per se is caused, so that the germination rate of seeds, the activity of plants and the yield of crops are seriously affected. With the use of a large amount of chemical fertilizers, the secondary salinization degree of soil is more and more serious, so that with the development of human society and the increase of population, the contradiction between the salinization of the soil and the increasing grain demand is generated. In general, there are about 953×10 worldwide 6 The irrigation area of hectares is affected by soil salinization; about 36X 10 in our country 6 Hectare saline-alkali soil, wherein the total area of the farmland of Xinjiang tank areas is 398.78 ten thousand hm 2 (without dry land), the saline-alkali soil area reaches 127.90 ten thousand hm 2 The saline-alkali soil accounting for 32.07 percent of the total cultivated land area in the irrigation area is a problem to be solved urgently. The method for improving the saline-alkali soil can be divided into water conservancy, agriculture, biology and chemical improvement measures. Flood irrigation is the most common treatment mode for improving saline-alkali soil, but has large water demand, and is not beneficial to being implemented in drought and low-water places; agricultural measures are multipurpose deep ploughing and deep ploughing, organic fertilizer application,Straw returning and the like, and the measures can improve the content of organic matters in soil; the chemical measures are that the soil quality is improved by using modifiers such as phosphoric acid, gypsum, wood vinegar and the like, but the use of the chemical modifiers can cause secondary pollution to the soil; biological measures are used for reducing soil surface stratification salts by increasing vegetation coverage and improving regional microclimate environments, such as by using saline-alkali tolerant crops and the like, or by using microbial methods. Rhizosphere microorganisms have been shown to promote plant growth in different saline-alkali soil environments. The screening of the saline-alkali tolerant growth promoting strain and the application of the strain in improving saline-alkali soil are very important. And the variety of the saline-alkali resistant growth promoting strain developed at present is relatively few.
Disclosure of Invention
In order to solve the technical problem of few varieties of saline-alkali resistant growth promoting strains developed at present, the invention provides a bacillus salicillusHalobacillussp, bachu85, the strain has multiple functions of salt and alkali tolerance, alkali reduction, phosphate dissolution, nitrogen fixation, IAA production, ACC deaminase production, siderophore production, catalase production, plant growth promotion and the like.
The object of the invention is achieved in the following way:
the strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation date of 2023, 3 and 28 days and a preservation number of CGMCC NO.26954.
The 16S rRNA gene sequence of the Bachu85 strain is shown as SEQ ID NO. 1.
The mutated structural gene is derived from the same species as the 16S rRNA gene sequence of Bachu85 strain.
The bacillus salicillusHalobacillussp, bachu85, wherein the tolerable salt concentration is 0 g/L-200 g/L, pH in the temperature range of 10-50 ℃ and the pH=7-11.
The Bachu85 strain and its progeny mutant and its derivative are applied in at least one of alkali reduction, organic phosphorus decomposition, nitrogen fixation, iron carrier production, hydrogen peroxidase production, low temperature growth and high temperature growth.
At least one application of the Bachu85 strain in saline-alkali soil improvement.
Said resistance toSalicornia camptothecaHalobacillusLiquid microbial agents, powder microbial agents and granular microbial agents prepared from sp, bachu85 and offspring, mutants and derivatives thereof;
the effective viable count of the liquid microbial inoculum is not less than 2.0 hundred million/mL, and the number of mold sundry fungus is not less than 2.0 multiplied by 10 6 The mixed bacteria rate is less than or equal to 7 percent per mL, and the pH value is 4.0-9.0;
the effective viable count of the powder microbial inoculum is not less than 2.0 hundred million/mL, and the number of mold sundry fungus is not less than 1.5 multiplied by 10 6 The bacterial contamination rate is less than or equal to 15 percent per mL, and the pH value is 4.0-9.0;
the effective viable count of the granular microbial agent is not less than 1.0 hundred million/mL, and the number of mold sundry fungus is not less than 1.2 multiplied by 10 6 The mixed bacteria rate is less than or equal to 20% per mL, and the pH is 4.0-9.0.
The microbial inoculum comprises one or more of the following auxiliary agents: glycerol, trehalose, amino acids, proteins, polysaccharides, sodium chloride, gamma-polyglutamic acid, sodium dodecylbenzenesulfonate, yeast extract, alcohol, potassium humate, and chitin; the microbial inoculum comprises one or more of the following solid carriers: peat, lignite, montmorillonite, silica, vermiculite, kaolin, diatomaceous earth, medical stone and alginate.
The microbial agent comprises one or more of a microbial agent and an inorganic fertilizer.
Compared with the prior art, the invention has the technical effects that: the invention provides a salt-tolerant bacillus caldusHalobacillusAnd sp.Bachu 85 strain, wherein the preservation number of the Bachu85 strain is CGMCCNO.26954. The Bachu85 strain has the functions of salt and alkali tolerance, alkali reduction, organic phosphorus decomposition, nitrogen fixation, IAA production, ACC deaminase production, catalase production, siderophore production, plant growth promotion and the like, wherein the IAA yield of the Bachu85 strain reaches 75.65 mug/mL. Therefore, the strain Bachu85 has better saline-alkali soil adaptability, saline-alkali soil improvement capability and plant growth promotion function.
Drawings
FIG. 1 is a phylogenetic tree of Bachu85 strain constructed based on the 16S rRNA sequence;
FIG. 2 is a graph of growth of Bachu85 strain;
FIG. 3 is a bar graph of colony diameters of Bachu85 strain at different salt concentrations;
FIG. 4 is a bar graph of growth of Bachu85 strain at different pH conditions;
FIG. 5 is a standard curve of IAA;
FIG. 6 is a graph of the results of IAA production experiments with Bachu85 strain;
FIG. 7 is a graph of experimental results of Bachu85 strain in decomposing organic phosphorus;
FIG. 8 is a graph of experimental results of nitrogen fixation by Bachu85 strain;
FIG. 9 is a graph of experimental results of Bachu85 strain siderophores;
FIG. 10 is a graph of experimental results of ACC deaminase produced by Bachu85 strain;
FIG. 11 is a graph showing the results of a growth-promoting experiment of Bachu85 strain on corn;
FIG. 12 is a graph showing the growth promotion results of Bachu85 strain on Arabidopsis thaliana;
FIG. 13 is a bar graph of the growth promoting effect of Bachu85 strain on Arabidopsis.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The invention provides a salt-tolerant bacillus caldus strain with salt toleranceHalobacillussp, bachu85 strain and application thereof, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 26954 in the year 3 and the month 28 of 2023.
HalobacillusIsolation, purification and identification of strains
1.Halobacillusseparation and purification of sp, bachu85 strains
Selecting a tamarix root soil sample in a karst region of Xinjiang, removing rhizosphere massive soil, gently shaking off rhizosphere soil, adding 5 g rhizosphere soil samples into a centrifuge tube filled with 45 mL sterilized deionized water, performing vortex vibration treatment for 2 min to enable microorganisms in the rhizosphere soil to be fully dispersed in the sterilized deionized water, and standing a soil mixed solution at room temperature for 3-5 min to form a soil suspension; sucking 1 mL soil suspension and adding 9The solution is prepared by sterilizing deionized water with a volume of 10 mL -1 The g/mL concentration suspension is diluted in sequence to obtain 10 -2 g/mL、10 -3 g/mL、10 -4 g/mL gradient suspension, and vortex shaking uniformly; taking 100 mu L of each diluted gradient of soil suspension, and coating the diluted gradient of soil suspension on a solid culture medium of beef extract peptone by using a sterilized coating rod, wherein each gradient is repeated three times; culturing in a constant temperature incubator at 30deg.C; and (3) picking single colonies, inoculating the single colonies into a beef extract peptone liquid culture medium for culture, and then carrying out streak pure culture, and repeating streaking for three times to obtain the pure cultured Bachu85 strain.
2. Identification of Bachu85 Strain
Gene amplification and alignment of Bachu85 Strain 16S rRNA
2 Xphanta Max Mix (p 515) of Novain Biolabs was selected, and PCR amplification was performed using the colony of the strain Bachu85 as a template, and the primers were bacterial universal primers 27F/1492R. The PCR amplification system was 25. Mu.L: primer 27F 1. Mu.L, primer 142R 1. Mu.L, 2 Xphanta Max Mix 12.5. Mu. L, DNA template 0.5. Mu. L, ddH 2 O10. Mu.L. The PCR amplification reaction procedure was set as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 2 min, cycle times of 35 times, extension at 72 ℃ for 5min again, and preservation at 4 ℃. The PCR amplified product is sent to the engineering Co.Ltd for sequencing, and the sequencing result is shown as a sequence table SEQ ID NO. 1. 16S rRNA and model species of Bachu85 strainHalobacillusdabanensis strain D-8 has a 99.86% similarity in gene sequence. Sequencing results of 16S rRNA were compared by NCBI database and phylogenetic tree was constructed as shown in FIG. 1. As can be seen from FIG. 1, the strain Bachu85 and model speciesHalobacillusdabanensis strain D-8 belongs to the smallest branch, has a relatively short evolutionary distance and is close to Chu Shi of Bacillus salicinusHalobacillusTrueperi) is far from evolution, and the two are not of the same species.
Morphological observations of Bachu85 Strain
The Bachu85 strain is subjected to three-area lineation on a solid beef extract peptone culture medium plate so as to separate single bacterial colonies, morphological observation is carried out on the single bacterial colonies, and the bacterial colonies of the Bachu85 strain on the beef extract peptone solid culture medium are round, transparent and milky, and have smooth and neat edges and no bulges.
The strain appeared purple after gram staining, indicating that Bachu85 strain belongs to gram positive bacteria.
Physiological and biochemical characteristics of Bachu85 Strain
The carbon source utilization condition of the strain Bachu85 is measured by utilizing a micro-fermentation tube of the Haibo biotechnology Co., ltd. And the result is shown in the table 1, the Bachu85 strain can grow by utilizing various substances as the only carbon source, and a foundation is provided for the production and application of the strain.
In conclusion, the strain Bachu85 is named as a combination of evolution analysis, morphological observation and physiological and biochemical characteristic measurement resultsHalobacillussp. Bachu 85。
TABLE 1 part of the physiological and biochemical indices of Bachu85 Strain
Note that: positive for "+"; negative of "-
Function of Bachu85 Strain
1. Saline-alkali tolerance function of Bachu85 strain
Growth curve determination of Bachu85 Strain
For further application, the growth curve of Bachu85 strain was determined.
Selecting single colony of Bachu85, culturing in beef extract peptone liquid medium, placing in constant temperature shaking table 30 deg.C, culturing at 180 r/min, measuring absorbance value of bacterial liquid at 600 nm with ultraviolet spectrophotometer, and waiting for OD of bacterial liquid 600 The value is 0.6-0.8; OD is set to 600 The Bachu85 bacterial liquid of the belonging bacterial strain with the value of 0.6-0.8 is inoculated into a beef extract peptone liquid culture medium with the inoculum size of 2 percent, the beef extract peptone liquid culture medium is placed in a constant-temperature shaking table for 30 ℃ and 180 r/min for culture, the absorbance value of the bacterial liquid at 600 nm is measured by an ultraviolet spectrophotometer every 4 h, and the liquid culture medium is used as a control group to draw a growth curve, as shown in figure 2. Bachu85 strain is in logarithmic growth phase in 4-16 h and is the strainThe production of the strain provides reference data.
Salt tolerance of Bachu85 Strain
Inoculating Bachu85 strain into sterile test tube containing beef extract peptone liquid medium, culturing at 30deg.C and 180 r/min, and measuring OD with ultraviolet spectrophotometer 600 Values. OD is then added again 600 Bacterial solutions with the value of 0.6-0.8 are respectively inoculated on solid culture media of beef extract peptone with the pH value of 9, namely 0 g/L, 20 g/L, 40 g/L, 60 g/L, 80 g/L, 100 g/L, 120 g/L, 140 g/L, 160 g/L, 180 g/L and 200 g/L, and solid culture media with the pH value of not adjusting and the salt concentration of 0 g/L for streaking culture, and are placed in a constant temperature incubator with the temperature of 30 ℃ for culture.
The growth state of the colonies was observed every 24 th h, and the diameter size of the strain was measured, and the results are shown in Table 2 and FIG. 3. The result shows that the Bachu85 strain has excellent salt tolerance, can normally grow under the condition of salt concentration of 0 g/L-200 g/L, and provides a foundation for the colonization and application of the strain in saline-alkali soil. The salt tolerance range of the Chu Shi bacillus salicillus is 100 g/L-200 g/L.
TABLE 2 salt tolerance results of Bachu85 Strain
Note that: "0 (-)" means that no NaCl is added and the pH is not adjusted; "0" means adjusting the pH with NaOH; "+" growth; "-" does not grow
Alkali-resistant function of Bachu85 Strain
Inoculating Bachu85 strain into sterile test tube containing beef extract peptone liquid medium, culturing at 30deg.C and 180 r/min, and measuring OD with ultraviolet spectrophotometer 600 Values. OD is set to 600 Bacterial solutions with the values of 0.6-0.8 are respectively inoculated into liquid culture mediums with the pH value of 7, pH value of 8, pH value of 9, pH value of 10, pH value of 11, pH value of 12 and pH value of 13 according to the inoculum size of 2 percent, and the liquid culture mediums are placed in a constant-temperature shaking table for 30 ℃ and 180 r/min for culture.
The absorbance of the bacterial liquid at 600 nm was measured at intervals of 24. 24 h by ultraviolet spectrophotometer, and the results are shown in Table 3 and FIG. 4, wherein the absorbance was obtained by repeating the steps three times at each time point with the liquid medium without bacteria as a control. The Bachu85 strain has excellent alkali resistance, can grow normally under the condition of pH value of 7-11, and provides a foundation for the colonization and application of the strain in saline-alkali soil. The alkali resistance range of Chu Shi bacillus salicillus is pH=9-pH=12.
TABLE 3 alkali resistance results of Bachu85 Strain
Note that: "+" growth; "-" does not grow
2. Alkali-reducing function of Bachu85 strain
OD is set to 600 Bachu85 bacterial liquid with the value of 0.6-0.8 is connected into beef extract peptone liquid culture medium with the pH value of 8, 9 and 10, and is placed into a constant-temperature shake culture box for culture at 30 ℃ and 180 r/min, 4 mL bacterial liquid is taken every 12 h, centrifugation is carried out at 4000 rpm for 5min, and the supernatant is taken and the pH value is measured by a pH meter. The alkali-reducing result of the Bachu85 strain is shown in Table 4, which shows that the Bachu85 strain has excellent alkali-reducing function, can effectively improve soil property and relieve stress caused by alkali on plants, thereby being effectively applied to improvement of saline-alkali soil and plant growth promotion. Chu Shi Bacillus salicinus has no alkali-reducing ability.
The alkali reduction rate was calculated according to the following formula:
η alkali =（pH Front part -pH Rear part (S) ）/pH Front part ×100%
Wherein: η (eta) Alkali The alkali reduction rate of the strain is reduced;
pH front part The pH value of the culture medium before inoculation;
pH rear part (S) The pH value of the culture medium after inoculation.
TABLE 4 alkali reduction results for Bachu85 Strain
3. IAA-producing function of Bachu85 Strain
The purified Bachu85 strain was inoculated into sterilized beef extract peptone medium containing 200 mg/L-tryptophan and cultured in a constant temperature shaking table at 30℃and 180 r/min. mu.L of the bacterial suspension was pipetted onto a white ceramic plate, 50. Mu.L of Salkowski developer was added, the white ceramic plate was kept at room temperature and protected from light for 30 min, and the color change was observed (if the color became red, it was indicated that the strain had IAA-producing function). The result shows that after Salkowski is added dropwise into the bacterial liquid of the Bachu85 strain, the color of the bacterial liquid in a white porcelain plate becomes red under the condition of light shielding at room temperature, and the preliminary indication that the Bachu85 strain has the function of producing IAA.
To determine the amount of IAA produced by the strain Bachu85, a standard curve of IAA was plotted in this section. Accurately weighing 10 mg IAA, dissolving with a small amount of absolute ethyl alcohol, then fixing the volume to 100 mL with distilled water to prepare 100 mug/mL IAA mother liquor, and then diluting to 10, 20, 30, 40 and 50 mug/mL standard liquor according to the proportion. Respectively taking the standard solutions 2 and mL, sequentially adding the standard solutions into 6 test tubes respectively, respectively adding 2 mL of Salkowski color development solution, and standing in a dark place for reaction for 30 min. The absorbance at record 530 nm is measured with a spectrophotometer, i.e., A 530 . Then in A 530 IAA concentrations are plotted on the abscissa and IAA standard curves are plotted, with the results shown in FIG. 5.
Inoculating the purified Bachu85 strain into a beef extract peptone liquid culture medium containing L-tryptophan, and placing the beef extract peptone liquid culture medium into a shaking table 180 r/min at the constant temperature of 30 ℃ for shake culture; then sucking the bacterial suspension of Bachu85 strain into a centrifuge tube, centrifuging for 10 min at 14000rcf, adding the supernatant into an equal volume of Salkowski chromogenic solution, standing in a dark place for reaction for 30 min, and measuring A 530 The IAA-producing concentration was calculated by regression equation of the standard curve, and the results are shown in table 5 and fig. 6. The quantitative experiment result shows that the IAA content in the Bachu85 fermentation liquid reaches the highest value, and each milliliter of the Bachu85 strain fermentation liquid contains 75.65 mug of IAA, so that the Bachu85 strain can produce a large amount of IAA, the IAA yield of Chu Shi bacillus salixidans is 35 mug/mL, and the IAA yield is obviously lower than that of the Bachu85 strain. IAA can alleviate the toxicity of saline-alkali stress to plants by regulating hormone levels in plants, thereby promoting plant growth.
TABLE 5 IAA production results of Bachu85 Strain
4. Phosphate solubilizing function of Bachu85 strain
Inoculating bacterial suspension of Bachu85 strain on Meng Jinna organophosphorus identification culture mediums, inoculating three parts on each culture medium, 15 mu L each part, placing in a 30 ℃ incubator for culturing for 7-9 d, and observing whether transparent rings appear around colonies. As a result, as shown in FIG. 7, the Bachu85 strain had a phosphate solubilizing circle diameter D of 14.18 mm, a colony diameter D of 11.35 mm and a D/D of 1.25 on Meng Jinna organophosphorus medium. The phosphate dissolving function of the Bachu85 strain can convert the phosphorus element which cannot be utilized by plants in soil into the available phosphorus element, so that sufficient phosphorus element nutrition is provided for the plants, and the growth of the plants is assisted; on the other hand, the phosphate solubilizing function of the Bachu85 strain can degrade organophosphorus pesticides remained in soil, so that the effect of repairing the soil is achieved, and the Chu Shi bacillus salicillus does not have the function of solubilizing organophosphorus.
5. Nitrogen fixation function of Bachu85 strain
Configuration of Abenta Bei Modan Medium (g/L): 0.2 Adding g of monopotassium phosphate, 10 g glucose, 0.2 g magnesium sulfate heptahydrate, 0.1 g calcium sulfate, 75 g sodium chloride, 5 g calcium carbonate and 16 g agar into 1L deionized water, adjusting pH=7.0-7.2, and sterilizing at 115 ℃ under high temperature and high pressure steam for 30 min.
OD is set to 600 Bachu85 bacterial suspension with the value of 0.3 is inoculated on an AWA Bei Modan solid culture medium, each culture medium is inoculated at three places, 15 mu L of bacterial liquid is spotted on each place, the culture medium is placed in a constant temperature incubator at 30 ℃ for inversion culture for 7-9 days, the growth condition of the Bachu85 bacterial strain is observed, the result is shown in figure 8, the Bachu85 bacterial strain can normally grow on the AWA Bei Modan culture medium, the Bachu85 bacterial strain has nitrogen fixation capacity, chu Shi bacillus salis does not have nitrogen fixation function, and the Bachu85 can improve soil fertility through the nitrogen fixation function and promote better growth of plants.
7. Siderophore function of Bachu85 strain
0.012 g CAS was dissolved in 10 mL deionized water and then combined with 2 mL 1 mmol/LFeCl 3 Uniformly mixing the solutions to obtain a solution a; dissolving 0.015 g of CTAB in 8 mL deionized water to obtain a solution b, and slowly adding the solution a into the solution b to obtain a dye liquor c; weighing Na 2 HPO 4 30 g，KH 2 PO 4 1.5 g， NaCl 2.5 g， NH 4 Cl 5 g, dissolved in 500 mL deionized water, gave 10 x MMq salt solution; 20 mL of 10X MMq salt solution is added into 150 mL deionized water, then 6.04 g of 1, 4-piperazine diethyl sulfonic acid is added, the pH value is regulated to be 6.8 by 1mol/L NaOH, and then 3.2 g agar powder is added to obtain a culture medium d; dye liquor c, culture medium d, 20% glucose and 1 mmol/L MgSO 4 ·7H 2 O、1 mmol/L CaCl 2 10% acid hydrolyzed casein is sterilized by high temperature and high pressure steam at 115 deg.C for 20 min. Taking the sterilized 1 mmol/L MgSO 4 ·7H 2 O 4 mL、1 mmol/L CaCl 2 0.2 And adding the mL, 10% acid hydrolyzed casein 6 mL and 20% glucose 2 mL into the culture medium d, and fully and uniformly mixing.
And adding a certain amount of CAS blue detection liquid into a complex culture medium consisting of acid hydrolyzed casein and other inorganic salts to prepare a blue detection plate. The rapid and convenient detection method does not depend on the structure of the siderophores, but detects the siderophores through chelate chromogenic reaction. In the presence of siderophores, the siderophores can react with Fe 3+ Forming stable chelate to make Fe 3 + From Fe 3+ And dye to form chelate to form stable chelate to make the detection board orange or red. Whether the strain produces pig iron carrier can be judged according to whether orange-red color-changing rings appear.
OD is set to 600 Bacterial suspension of Bachu85 strain with the concentration of 0.3 is inoculated on a CAS solid culture medium, the solid culture medium is placed in a constant temperature incubator at 30 ℃ for culturing 3 d, the growth condition of the Bachu85 strain is observed, if orange or orange-red transparent rings appear, the strain is proved to produce pig iron carrier, as shown in figure 9, the bacterial strain Bachu85 has the function of producing the pig iron carrier, while Chu Shi bacillus salicinus has no description of the function of producing the pig iron carrier, and Bachu85 can produce insoluble Fe 3+ Is converted into Fe which is easy to be absorbed by plants 2+ Thereby promoting plant growth.
8. ACC-producing deaminase function of Bachu85 Strain
Preparing ADF culture medium: accurately weigh KH 2 PO 4 4.0 g、Na 2 HPO 4 6.0 g、MgSO 4 ·7H 2 O0.2 g, glucose 2.0 g, gluconic acid 4 mL, citric acid 2.0 g, ACC 3.0-5.0 mmol (added after sterilization), agar powder 15 g, added into 1L deionized water, pH=7.2, and sterilized at 115 ℃ for 30 min.
OD is set to 600 Bacterial suspension of Bachu85 strain with the concentration of 0.3 is inoculated on ADF solid culture medium, placed in a constant temperature incubator, and cultured in an inverted mode at the temperature of 30 ℃ for 7 d, and the growth condition of the Bachu85 strain is observed, if the Bachu85 strain can normally grow, the Bachu85 strain has the function of producing ACC deaminase. As a result, as shown in FIG. 10, bachu85 strain was able to grow normally on ADF medium, demonstrating that Bachu85 strain has ACC deaminase-producing function. Ethylene has an inhibition effect on plant growth, ACC is a precursor substance of ethylene, and ACC deaminase generated by Bachu85 strain can decompose ACC into ammonia, so that the inhibition effect of ethylene on plants is relieved, and a nitrogen source is provided for plant growth. The strain Bachu85 can thus help plants resist the stress of ethylene and promote plant growth.
9. Catalase-producing function of Bachu85 strain
The part is determined by utilizing a catalase kit of Haibo biotechnology Co., ltd, bachu85 strain is inoculated into LB liquid culture medium until bacterial liquid OD 600 And (3) dripping the fungus dipped with a glass rod into a clean glass slide or test tube to obtain 0.6-0.8, then adding 1-2 drops of 3% hydrogen peroxide for observation, and judging that the bacterial strain to be tested generates catalase if bubbles are generated. The result shows that the Bachu85 strain generates a large amount of bubbles after hydrogen peroxide is dripped, and the Bachu85 strain has the capability of generating catalase, and the Chu Shi bacillus salicillus does not generate catalase. The excessive hydrogen peroxide can cause damage to plants and influence the growth of the plants, and the catalase can be used as a hydrogen peroxide scavenger to reduce the damage of the hydrogen peroxide to the plants and finally promote the growth of the plants.
Chu Shi the salt tolerance range of the bacillus calsalis is 100 g/L-200 g/L, the alkali resistance range is pH=9-12, the functions of nitrogen fixation, organic phosphorus decomposition, siderophore production and catalase production are not provided, the IAA yield is 35 mug/mL at maximum, and the potassium decomposition and ACC deaminase production can be realized. The Bachu85 salt tolerance range is 0 g/L-200 g/L, the alkali resistance range is pH=7-pH=11, the system has the functions of nitrogen fixation, organic phosphorus decomposition, ACC deaminase production, siderophore production and catalase production, the IAA yield is 75.65 mug/mL at maximum, the potassium decomposition function is avoided, and the system evolution tree of the 16S rRNA sequence comparison result shows that: chu Shi the evolution distance between the bacillus salicillus and Bachu85 is far; thus, bachu85 and Chu Shi bacillus salicillus are different, and belong to different species.
Growth promoting function of strain on corn
Preparing a bacterial suspension: inoculating Bachu85 strain into beef extract peptone liquid culture medium, placing in constant temperature shaking table 30 deg.C, culturing at 180 r/min, diluting culture solution with sterile water to thallus concentration of 10 8 CFU/mL was used as bacterial suspension.
Selecting complete and full corn seeds, washing off red coats of the seeds by deionized water, sterilizing for 0.5-1 min by using 95% alcohol, and soaking for 15min by using 0.1% sodium hypochlorite solution; then cleaning the seeds with sterilized deionized water for 4 times, finally filling the seeds into a beaker, adding sterile water to immerse the corn seeds, and accelerating germination under the condition of normal temperature and darkness for one night.
Uniformly mixing nutrient soil and vermiculite according to a ratio of 1:1, and sterilizing for 3 times at 121 ℃ for 20 min under high temperature and high pressure steam; using NaHCO 3 Mixing with neutral salt according to the mass ratio of 1:1 to obtain 15 g/kg and 30g/kg salt solution; the salt solution is mixed with the sterilized soil uniformly. Sowing the treated seeds in small black pots, wherein each pot is used for sowing 5 seeds, and the sowing depth is about 1 cm; 10 replicates were set for each group; when two leaves of corn grow out, 5 mL of the prepared bacterial suspension is applied to each experimental group, 5 mL of sterilized deionized water is applied to the control group, 5 mL of the prepared bacterial suspension is applied to the experimental group every other week, and 5 mL of sterilized deionized water is applied to the control group; after 14 and d, the fresh weight above the ground and the fresh weight below the ground are measured, and the stem is thick and the plant height is high. The results showed that Bachu85 strain was inoculated under conditions of 0g/kg, 15 g/kg and 30g/kgThe experimental results are shown in Table 6 and FIG. 11, wherein A, B, C represents stress treatment conditions of 0g/kg, 15 g/kg and 30g/kg, respectively. Under the condition of 0g/kg of treated soil, the overground fresh weight, the underground fresh weight and the plant height growth indexes of the plants treated by inoculating Bachu85 strain are respectively increased by 123.9305%, 71.5251% and 17.4%, and the growth is obvious and extremely obvious difference is achieved.
TABLE 6 growth-promoting experiment results of Bachu85 Strain
Growth promoting effect of Bachu85 strain on Arabidopsis thaliana under saline-alkali stress
1/2 MS solid medium for 5 saline-alkali stress conditions was prepared: (1) ph=5.8, no NaHCO 3 And NaCl; (2) ph=8, no NaHCO 3 And NaCl; (3) pH=8, add 2 mmol/L NaHCO 3 The method comprises the steps of carrying out a first treatment on the surface of the (4) ph=8, plus 100 mmol/L NaCl; (5) pH=8, add 2 mmol/L NaHCO 3 And 100 mmol/L NaCl. The pH of the culture medium is regulated by 1mol/L NaOH, and the culture medium is sterilized at high temperature and high pressure for 30 min at 115 ℃.
Five conditions of 1/2 MS medium were plated separately, after the medium was thoroughly cooled, the seeds of Arabidopsis that had been vernalized at 4℃were spotted on 1/2 MS medium 6.0 cm from the bottom of the dish while the Bachu85 strain was inoculated at the bottom, and three biological replicates were performed for each treatment condition. Then, the culture dish is vertically placed (one end of the inoculated Arabidopsis seeds is upwards) in an illumination incubator at a temperature of 22 ℃, illumination intensity of 12000 Lux, photoperiod of 16 h/8 h and relative humidity of 50 percent for 10 days, and physiological indexes such as root length, lateral root number, fresh weight, leaf number and the like are measured. The results are shown in FIG. 12 and FIG. 13, and it can be intuitively seen from FIG. 12 that Bachu85 inoculation under different conditions has a remarkable promoting effect on root length, fresh weight and leaf number of Arabidopsis thaliana, wherein the condition of a culture medium is pH=5.8, and NaHCO is not added 3 And NaCl; b culture medium at pH=8, no NaHCO added 3 And NaCl; c Medium conditions were pH=8, 2 mmol/L NaHCO was added 3 The method comprises the steps of carrying out a first treatment on the surface of the d medium conditions were ph=8, plus 100 mmol/L NaCl; e cultivatingThe culture medium condition is pH=8, and 2 mmol/L NaHCO is added 3 And 100 mmol/L NaCl. At ph=8, no NaHCO was added 3 And NaCl and ph=8, add NaHCO 3 Under the two stress conditions, the Bachu85 strain has remarkable promotion effect on the lateral root number of the arabidopsis, and after 100 mmol of NaCl is added, the fresh weight and the leaf number of the arabidopsis can be increased by inoculating the Bachu85 strain.
FIG. 13 is a bar graph of the growth promoting results of Bachu85 strain on Arabidopsis, wherein A, B, C, D is the effect on main root length, lateral root number, fresh weight and leaf number, respectively, of Arabidopsis. In fig. 13, the medium a was conditioned at ph=5.8 without NaHCO addition 3 And NaCl; b culture medium at pH=8, no NaHCO added 3 And NaCl; c Medium conditions were pH=8, 2 mmol/L NaHCO was added 3 The method comprises the steps of carrying out a first treatment on the surface of the d medium conditions were ph=8, plus 100 mmol/L NaCl; e Medium conditions were pH=8, 2 mmol/L NaHCO was added 3 And 100 mmol/L NaCl. From the bar graph, it can be seen that the Bachu85 strain inoculated under different conditions has obvious growth promoting effect on arabidopsis seedlings. At ph=8, no NaHCO was added 3 And NaCl and pH=8, 2 mmol/L NaHCO is added 3 In addition, after 100 mmol of NaCl is added, bachu85 is inoculated to increase the fresh weight of Arabidopsis thaliana and the leaf number of Arabidopsis thaliana.
In conclusion, the saline-alkali tolerant bacillus caldus Bachu85 can obviously promote the growth of arabidopsis seedlings.
Microbial inoculum containing bacillus salicillus Bachu85
By means ofHalobacillusThe liquid microbial inoculum of the bacillus caldus Bachu85 provided in the steps of separating and purifying the bacillus caldus Bachu85 strain comprises one or more of inorganic fertilizer and microbial inoculum. The auxiliary agent in the Bachu85 liquid microbial inoculum comprises one or more of glycerol, trehalose, amino acid, protein, polysaccharide, sodium chloride, gamma-polyglutamic acid, sodium dodecyl benzene sulfonate, yeast extract, alcohol, potassium humate and chitin. The solid carrier comprises one or more of peat, lignite, montmorillonite, silica, vermiculite, kaolin, diatomite, medical stone and alginate.
The invention providesBacillus salicillusHalobacillussp, bachu85 is separated from rhizosphere soil of salix chinensis, has stronger saline-alkali resistance and provides a foundation for the colonization of the strain. The Bachu85 strain has the characteristics of reducing alkali, degrading organic phosphorus, fixing nitrogen, producing IAA, producing ACC deaminase, producing catalase, siderophore and the like, can effectively improve the soil microenvironment of plant root systems, provide nutritional ingredients for plant growth, reduce the stress of the saline alkali on plants, and finally achieve the function of promoting the plant growth. Therefore, the strain Bachu85 has important application value in the aspects of improving saline-alkali soil, improving the saline-alkali stress resistance of plants, promoting plant growth and the like.
The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.
Claims (9)
1. Saline-alkali tolerant bacillus camptothecaHalobacillussp, bachu85, wherein the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2023, 3 and 28 days and a preservation number of CGMCC NO.26954.
2. The Bachu85 strain of claim 1, wherein the Bachu85 strain has a 16S rRNA gene sequence as shown in SEQ ID No. 1.
3. A mutated structural gene naturally obtained from the 16S rRNA gene sequence of Bachu85 strain according to claim 1 or 2, characterized in that the mutated structural gene originates from the same species as the 16S rRNA gene sequence of Bachu85 strain.
4. The bacillus salixidans according to claim 1 or 2Halobacillussp, bachu85, characterized in that: the bacillus salicillusHalobacillussp, bachu85, wherein the tolerable salt concentration is 0 g/L-200 g/L, pH in the temperature range of 10-50 ℃ and the pH=7-11.
5. The Bachu85 strain and its progeny mutant and its derivatives as claimed in claim 1-3, wherein the Bachu85 strain and its derivatives are used for at least one of alkaline reduction, organic phosphorus decomposition, nitrogen fixation, siderophore production, catalase production, low temperature growth and high temperature growth.
6. The Bachu85 strain of any of claims 1-3 for use in at least one of saline-alkali soil improvement.
7. Use of the salt-tolerant bacillus caldus according to any one of claims 1 to 3HalobacillusLiquid microbial agents, powder microbial agents and granular microbial agents prepared from sp, bachu85 and offspring, mutants and derivatives thereof;
the effective viable count of the liquid microbial inoculum is not less than 2.0 hundred million/mL, and the number of mold sundry fungus is not less than 2.0 multiplied by 10 6 The mixed bacteria rate is less than or equal to 7 percent per mL, and the pH value is 4.0-9.0;
the effective viable count of the powder microbial inoculum is not less than 2.0 hundred million/mL, and the number of mold sundry fungus is not less than 1.5 multiplied by 10 6 The bacterial contamination rate is less than or equal to 15 percent per mL, and the pH value is 4.0-9.0;
the effective viable count of the granular microbial agent is not less than 1.0 hundred million/mL, and the number of mold sundry fungus is not less than 1.2 multiplied by 10 6 The mixed bacteria rate is less than or equal to 20% per mL, and the pH is 4.0-9.0.
8. The microbial inoculant of claim 7, wherein the microbial inoculant comprises one or more of the following adjuvants: glycerol, trehalose, amino acids, proteins, polysaccharides, sodium chloride, gamma-polyglutamic acid, sodium dodecylbenzenesulfonate, yeast extract, alcohol, potassium humate, and chitin; the microbial inoculum comprises one or more of the following solid carriers: peat, lignite, montmorillonite, silica, vermiculite, kaolin, diatomaceous earth, medical stone and alginate.
9. The microbial agent of claim 7, wherein the microbial agent comprises one or more of a microbial agent and an inorganic fertilizer.
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