CN110117560B

CN110117560B - Rhizosphere growth-promoting bacterium for enhancing salt tolerance of crops, microbial fertilizer and application thereof

Info

Publication number: CN110117560B
Application number: CN201910359879.1A
Authority: CN
Inventors: 张瑞福; 陈小娟; 李盼; 刘云鹏; 徐志辉; 邵佳慧
Original assignee: Nanjing Agricultural University; Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Nanjing Agricultural University; Institute of Agricultural Resources and Regional Planning of CAAS
Priority date: 2019-04-30
Filing date: 2019-04-30
Publication date: 2021-01-05
Anticipated expiration: 2039-04-30
Also published as: CN110117560A

Abstract

The invention discloses a rhizosphere growth-promoting bacterium for enhancing salt tolerance of crops, a microbial fertilizer thereof and application thereof. The Bacillus licheniformis (Bacillus paralicheniformis) T1-8 provided by the invention has the preservation number of CGMCC No. 17335. The strain is inoculated to the rhizosphere of potted corn and wheat, and the growth indexes of corn and wheat can be improved under the condition of salt stress. The microbial organic fertilizer is prepared by uniformly mixing the fermented bacterial liquid of the strain and common cow dung, pig dung and straw, composting and performing secondary fermentation, and can improve the corn yield of saline-alkali soil and improve the physical and chemical properties of soil to a certain extent. The invention has important significance for developing functional microbial organic fertilizers (enhancing salt stress resistance of crops and improving the crop yield of saline-alkali soil).

Description

Rhizosphere growth-promoting bacterium for enhancing salt tolerance of crops, microbial fertilizer and application thereof

Technical Field

The invention relates to the technical field of agricultural biological product production, in particular to a rhizosphere growth-promoting bacterium for enhancing salt tolerance of crops, a microbial fertilizer and application thereof.

Background

At present, a large amount of cultivated land in the world is affected by salt damage, excessive salt in soil can inhibit the growth of crops and reduce the yield of the crops, the salinization of the soil is an important problem in global agricultural development, and irrigation water and seawater containing trace sodium chloride are the main reasons for increasing the salt content in the soil of the farmland. The increase of the NaCl content in the soil can reduce the water absorption capacity of the plants, and the salt stress can influence the germination of the seeds through ways of osmotic stress, ion toxicity, oxidative damage, enzyme activity inhibition and the like. The known methods for enhancing the salt tolerance of plants are various, salt in the environment can be reduced by cultivating salt tolerance plant varieties or constructing transgenic plants, and also by improving the environment, such as modifying the environment structure and leading water to wash a salt-affected area, but the transgenic crops are low in public acceptance at present, the engineering of modifying the environment structure is large, plant rhizosphere growth-promoting bacteria can be another mode for effectively enhancing the salt tolerance of the plants, and the defects of the two methods are overcome. Under salt stress, the rhizosphere growth-promoting bacteria can reduce the stress injury of plants by inducing the tolerance of an induction system. Common rhizosphere growth-promoting bacteria for enhancing the salt stress tolerance of plants comprise Chryseobacterium, Pseudomonas mendocina, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus subtilis and the like. Under the stress of salt damage, the rhizosphere growth-promoting bacteria can enhance the abiotic stress resistance of plants by increasing nutrient absorption rate, reducing active oxygen damage, generating phytohormones, generating VOCs, increasing osmotic protective agents, regulating plant ethylene and the like.

The microbial fertilizer refers to a specific product containing living microorganisms, and the fertilizer effect is brought by the life activities and metabolites of the microorganisms. With the increase of the usage amount of chemical fertilizers and pesticides in China, a series of ecological and economic problems are brought, such as soil hardening, acidification, aggravation of soil-borne diseases and water eutrophication, meanwhile, although the usage amount of the chemical fertilizers is increased, the utilization efficiency is lower and lower, the yield of crops is increased slightly, the production cost is increased, the economic income of farmers is reduced, and the production enthusiasm is stricken seriously, so that the development of agriculture is limited. The microbial fertilizer can increase the utilization rate of chemical fertilizers, improve the quality of crops, improve the soil structure, increase the stress resistance and disease resistance of the crops and improve the yield of the crops, and is a novel green, environment-friendly and nontoxic fertilizer. The development of functional microbial organic fertilizer (enhancing the salt stress resistance of crops and improving the crop yield in saline-alkali soil) is a feasible way.

Disclosure of Invention

The invention aims to develop and develop a microbial organic fertilizer capable of improving the salt stress resistance of saline-alkali soil crops, improving the yield of the saline-alkali soil crops and improving the soil physicochemical property of the saline-alkali soil to a certain extent aiming at the actual problem that the saline-alkali soil crops have low yield and the land is difficult to utilize in agricultural production.

In order to solve the technical problems, the invention firstly provides Bacillus licheniformis (Bacillus Paralicheniformis) T1-8.

The Bacillus licheniformis (Bacillus paracheniformis) T1-8 provided by the invention has been preserved in China general microbiological culture Collection center (CGMCC for short; No. 3 of Beijing university Hokko No.1 of Shangyang district, China academy of sciences microbial research, and zip code 100101) in 03 and 15 days in 2019, and the preservation number is CGMCC No. 17335.

Bacillus paracasei (Bacillus paraceriniformis) T1-8 is simply called Bacillus T1-8.

The fermentation product of the bacillus T1-8 also belongs to the protection scope of the invention.

The fermentation product of the bacillus T1-8 is obtained by inoculating the bacillus T1-8 into a liquid fermentation culture medium for fermentation culture;

the liquid fermentation medium consists of solute and solvent; the solute and the concentration thereof in the liquid fermentation medium are as follows: 4-6g/100ml of molasses, 0.7-0.8g/100ml of soybean meal, 0.4-0.6g/100ml of sodium chloride, 0.05-0.15g/100ml of monopotassium phosphate, 0.05-0.15g/100ml of calcium chloride, 0.05-0.15g/100ml of magnesium sulfate, 0.04-0.06g/100ml of ferrous sulfate, 0.04-0.06g/100ml of zinc sulfate and 0.04-0.06g/100ml of manganese sulfate; the solvent is water.

In the most preferred liquid fermentation media described in the examples of the present invention, the solutes and their concentrations in the liquid fermentation media are: 5g/100ml of molasses, 0.72g/100ml of soybean meal, 0.5g/100ml of sodium chloride, 0.1g/100ml of monopotassium phosphate, 0.1g/100ml of calcium chloride, 0.1g/100ml of magnesium sulfate, 0.05g/100ml of ferrous sulfate, 0.05g/100ml of zinc sulfate and 0.05g/100ml of manganese sulfate.

The pH of the liquid fermentation medium was 7.5.

In the fermentation culture, Bacillus T1-8 is inoculated into the liquid fermentation medium, and the initial bacteria content in the fermentation system can be 5 × 10⁷One per ml.

The method for realizing the initial bacteria content specifically comprises the following steps: inoculating bacillus T1-8 into LB liquid culture mediumCulturing to obtain bacterial suspension (the bacterial concentration in the bacterial suspension is 10)⁹one/mL); transferring the bacterial suspension into the liquid fermentation medium, wherein the inoculation amount is 5% (volume percentage content).

During the fermentation culture, the fermentation temperature can be 35 ℃, the rotation speed can be 180rpm, and the fermentation time can be 48 hours.

The fermentation product can be a fermentation system obtained by the fermentation.

The number of bacteria or spores of the Bacillus T1-8 in the fermentation product is 10¹⁰More than one/mL.

The invention also protects the application of the bacillus T1-8 or the fermentation product, which is (a1) or (a 2):

(a1) relieving the damage of salt stress to plants;

(a2) improving the salt stress resistance of the plants.

In the application, (a1) or (a2) can be embodied in that the growth indexes (plant height, overground fresh weight and root length) of the plants subjected to salt stress are improved after being inoculated with the bacillus T1-8 compared with the growth indexes of the plants which are also subjected to salt stress but are not inoculated with the bacillus T1-8.

In said use, (a1) or (a2) can be embodied in that the plants which are subjected to salt stress, after inoculation with Bacillus T1-8, have an increased Total Soluble Sugars (TSS) and/or an increased Peroxidase (POD) activity and/or a reduced Malondialdehyde (MDA) content and/or hydrogen peroxide (H) content in comparison with plants which are likewise subjected to salt stress but are not inoculated with Bacillus T1-8₂O₂) Reduced content and/or reduced superoxide dismutase (SOD) activity and/or reduced Catalase (CAT) activity.

The invention also protects the application of the bacillus T1-8 or the fermentation product in preparing microbial fertilizer.

The invention also protects a microbial fertilizer, and each gram of the microbial fertilizer contains 5 multiplied by 10⁸-2×10⁹The bacillus T1-8.

The microbial fertilizer prepared by the embodiment of the invention also meets the following conditions: (1) the mass percentage of the organic matter is more than 45%; (2) the sum of the mass percentage of the nitrogen, the phosphorus and the potassium is more than 5 percent; (3) the water content is 50-60%; (4) the pH value is 6.5 to 7.5. The (2) is embodied that the mass percentage of nitrogen is more than 1.5%, the mass percentage of phosphorus is more than 2.5%, and the mass percentage of potassium is more than 1%.

The invention also provides a microbial fertilizer, and the preparation method comprises the following steps: and mixing the fermentation product of the bacillus T1-8 with an organic material for secondary solid fermentation to obtain the microbial fertilizer.

The organic material can be any commercially available common organic fertilizer, and can also be any decomposed organic materials such as livestock and poultry manure, straws and the like.

The organic material may be an organic material satisfying the following conditions: (1) the mass percentage of the organic matter is more than 45%; (2) the sum of the mass percentage of the nitrogen, the phosphorus and the potassium is more than 5 percent; (3) the pH value is 6.5 to 7.5. The (2) is embodied that the mass percentage of nitrogen is more than 1.5%, the mass percentage of phosphorus is more than 2.5%, and the mass percentage of potassium is more than 1%.

Before the secondary fermentation, the organic materials are supplemented with water until the water content reaches 50-65%.

The secondary solid fermentation meets the following conditions: (a) the fermentation temperature is 30-50 ℃; (b) the water content is kept between 50 and 65 percent in the fermentation process; (c) turning the pile in the middle of fermentation.

The number of days of the secondary solid fermentation may be specifically 7 days. When the fermentation days are 7 days, one pile turning can be carried out on the fourth day.

In the secondary solid fermentation, 30L of the fermentation product can be added into each ton of organic materials. The 30L fermentation product may specifically contain 3 × 10¹⁴-5×10¹⁴The bacillus T1-8.

The organic materials adopted in the embodiment of the invention are decomposed cow dung, pig dung and straw mixed compost, wherein the total nitrogen content is 1.56%, the total phosphorus content is 2.52%, the total potassium content is 1.32%, the organic matter content is 48.51%, the water content is 28.26, and the pH value is 6.8.

In the embodiment of the invention, the specific operation method of the secondary solid fermentation is as follows: mixing an organic material with a fermentation product of bacillus T1-8 (30L of the fermentation product can be added into each ton of the organic material), performing secondary solid fermentation in a ventilated and cool place, turning over the pile in the fourth day, keeping the fermentation temperature at 30-50 ℃, controlling the water content of a fermentation system at 50-60%, and ending the fermentation after 7 days to obtain the microbial organic fertilizer.

The microbial fertilizer prepared by the method contains 5 x 10/g⁸-2×10⁹The bacillus T1-8.

The invention also protects the application of any one of the microbial fertilizers, which is at least one of the following (b1) - (b 5):

(b1) relieving the damage of salt stress to plants;

(b2) improving the salt stress resistance of plants;

(b3) as a fertilizer for plants in saline and alkaline land;

(b4) the plant yield of the saline-alkali soil is improved;

(b5) the physicochemical property of the saline-alkali soil is improved.

In the step (b5), the physicochemical properties of the saline-alkali soil can be embodied in that the organic matter content of the soil is increased and/or the pH is reduced and/or the salt content of the soil is reduced after the microbial fertilizer is used compared with the common fertilizer.

Any of the above plants may be dicotyledonous or monocotyledonous.

The plant may be a graminaceous plant.

The plant may be wheat or corn.

The invention provides a strain capable of improving salt stress resistance of corn, which is inoculated to the rhizosphere of potted corn and wheat, so that the growth indexes (plant height and overground fresh weight) of the corn and the wheat can be improved under salt stress, the Total Soluble Sugar (TSS) and Peroxidase (POD) activities in corn leaves can be improved, and simultaneously the contents of Malondialdehyde (MDA) and hydrogen peroxide (H2O2) and the activities of superoxide dismutase (SOD) and Catalase (CAT) in the corn leaves can be reduced. In addition, the strain T1-8 also has the growth promoting capabilities of producing ammonia, decomposing inorganic phosphorus, producing siderophin, producing IAA and the like, and is a rhizosphere growth promoting bacillus. The microbial organic fertilizer is prepared by uniformly mixing the fermented bacterial liquid of the strain and common cow dung, pig dung and straw for composting and then performing secondary fermentation, and has the following advantages:

1. the microbial organic fertilizer provided by the invention is specially used for improving the corn yield of saline-alkali soil and improving the physical and chemical properties of the soil to a certain extent, and not only has the effect of increasing the yield, but also can improve the soil;

2. the effective bacterial strain in the microbial organic fertilizer provided by the invention is derived from saline-alkali soil, has adaptability to saline-alkali environment, has the capabilities of producing ammonia, decomposing inorganic phosphorus, producing siderophil and the like, is negative in hemolytic reaction, and does not harm human;

3. the main raw materials of the culture solution required by the early-stage liquid fermentation of the microbial organic fertilizer provided by the invention are molasses and soybean meal, the cost of the raw materials is low, and the utilization efficiency of the materials is improved. Under the optimal fermentation condition, the effective bacteria quantity or spore content in the bacteria liquid can reach the required quantity in a shorter fermentation period;

4. the carrier organic material used by the microbial organic fertilizer provided by the invention is common cow dung, pig dung and straw mixed compost, does not need specific organic fertilizer bearing, can be normally used after secondary fermentation, and has common raw materials as the carrier, easy obtainment and low cost.

Drawings

FIG. 1 shows the results of measuring the root length of some strains in the preliminary screening of the strains in example 1.

FIG. 2 shows the results of phenotypic observations of maize and wheat during the rescreening (hydroponics) of the strains of example 1.

FIG. 3 shows the results of phenotypic observations of maize and wheat during the rescreening (soil culture) of the strain of example 1.

FIG. 4 shows the results of the detection of the correlation indicators in example 2.

FIG. 5 shows the results of the fermentation results under different experimental conditions of the single factor experiment in example 5.

FIG. 6 shows the results of the fermentation performance measurements under different conditions of the orthogonal experiment in example 5.

FIG. 7 is an observation of maize phenotype under different fertilization conditions in example 7.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The corn variety is Beijing sweet and glutinous: can be purchased from agricultural academy of sciences in Jiangsu province and is recorded in the document 'Wangjianhuang, Chengwei, Lihero billows, new variety of high-quality purple black waxy corn and main point of high-yield cultivation [ J ]. China breed, 2008(7): 79-80.'; the public is also available from research institutes of agricultural resources and agricultural divisions of the Chinese academy of agricultural sciences.

Wheat variety Bainong dwarf 58: is commercially available from agricultural academy of sciences in Henan province and is described in the document "Lexue, Lexue Hui, Qishang Red. New wheat variety Bainong SHO 58 characteristic features and high yield cultivation essence [ J ]. agricultural science and technology Communication 2006(9): 24-25.", the public also can be obtained from the institute of agricultural resources and agricultural divisions of the Chinese agricultural academy of sciences.

Corn variety constant source number one: commercially available from seed company junkerchien.

Example 1 sample Collection and Strain screening

First, sample collection and strain isolation

Collecting saline-alkali soil all over the country, carrying out pot experiment, planting the corn variety Jing sweet and glutinous rice on the soil, watering normally, and harvesting corn plants after one month. Respectively leaching soil suspensions of rhizosphere soil, diluting and coating the soil suspensions on an LB solid culture medium after water bath at 85 ℃ for 15min, culturing overnight at 37 ℃, respectively separating and purifying strains on a flat plate, and co-screening to obtain bacillus 124 strains.

Second, preliminary screening of the strains

The strains to be detected are as follows: 124 selected bacillus strains in the first step.

1. The surface of the seed of the maize variety Jing sweet and purple glutinous rice is sterilized and then placed in a sterilized culture dish (sterile filter paper wetted by sterile water is placed at the bottom of the culture dish), and vernalization is carried out for 3 days at 4 ℃ in a dark place.

2. After step 1 was completed, the germinated seeds were placed in a round plastic basket, 30 seeds were placed on each small basket, the plastic basket was placed on a 1.6L small bucket, about 1.4L of water was added until contacting the seeds, and the mixture was incubated at 25 ℃ for 4 days with water changed once a day with shading with a black shading film.

3. After step 2, the black light-shielding film is removed, the culture is continued for a week at 25 ℃, and water is changed every morning and evening.

4. After completion of step 3, the water in the keg was replaced with 1/4MS nutrient solution containing 140mM NaCl, and at the same time, the roots of the corn were inoculated with 10ml of 1/4MS nutrient solution containing the strain to be tested (concentration of the strain to be tested in 1/4MS nutrient solution was 10)⁷one/mL), incubated at 25 ℃ for 7 days.

3 replicates were set for each strain tested.

A control (Mock control) was set up using 1/4MS nutrient solution instead of 1/4MS nutrient solution with 140mM NaCl and without inoculation of the strain.

A control (Salt control) without inoculated strain was set.

5. And (4) after the step 4 is completed, measuring the plant height, the fresh weight of the overground part and the root length of the corn.

According to the statistical results of plant height, fresh weight of overground parts and root length, 2 strains with good effect are preliminarily screened, wherein the effect of the strain T1-8 is the best.

The results of partial root length measurements are shown in FIG. 1. In FIG. 1, ck100 is the statistics for root length of a control (Salt control) not inoculated with a strain, and the remainder are the statistics for root length of maize inoculated with different strains.

Thirdly, re-screening of bacterial strain (hydroponics)

The strains to be detected are as follows: and step three, 2 strains which are screened primarily.

1. The surfaces of the seeds of the maize variety Jing sweet and purple glutinous and the seeds of the wheat variety Bainong dwarf 58 are sterilized and then are placed in a sterilized culture dish (sterile filter paper wetted by sterile water is placed at the bottom of the culture dish), and are vernalized in a dark place for 3 days at 4 ℃.

4. After step 3 is completed, the water in the keg is replaced by 1/4MS nutrient solution containing NaCl, and at the same time, the root of corn or wheat is inoculated with 10ml of 1/4MS nutrient solution containing the strain to be tested (the concentration of the strain to be tested in 1/4MS nutrient solution is 10)⁷one/mL), incubated at 25 ℃ for 7 days.

In step 4, the corn group changes water into 1/4MS nutrient solution containing 140mM NaCl; the wheat group replaced water with 1/4MS nutrient solution containing 180mM NaCl.

3 replicates were set for each strain tested.

A control (Mock control) was set up using 1/4MS nutrient solution instead of 1/4MS nutrient solution with NaCl and without inoculation of the strain.

A control (Salt control) without inoculated strain was set.

According to the statistical result, the maize and wheat inoculated with the strain T1-8 have the best growth condition, and the maize and wheat inoculated with the strain T1-8 have the plant height and the fresh weight of the overground part which are all obviously higher than those of the control without the strain T1-8.

The observation of the maize and wheat phenotypes of the inoculated strain T1-8 is shown in FIG. 2. In FIG. 2, A is corn and B is wheat.

Thirdly, re-screening of the strain (earth culture)

1. Respectively sterilizing the surfaces of the seeds of the maize variety Jing sweet and purple glutinous and the seeds of the wheat variety Bainong dwarf 58, placing the sterilized seeds and the sterilized seeds in a sterilized culture dish (sterile filter paper wetted by sterile water is placed at the bottom of the culture dish), and vernalizing the seeds at 4 ℃ in a dark place.

2. After the step 1 is completed, the germinated seeds are placed in 700mL plastic cups with holes at the bottoms (1 kg of soil is filled in each plastic cup), 3 corn seeds and 10 wheat seeds are transplanted in each plastic cup respectively, the plastic cups are cultured for 10 days at 25 ℃, and 50mL of deionized water is poured every other day.

3. After step 2 was completed, 10mL of 1/4MS nutrient solution containing the test bacteria (to give a concentration of 10 bacteria in soil) was added to each plastic cup⁸Per g soil), continuously culturing for 10 days with the culture condition unchanged, and pouring 50mL of deionized water containing 200mM NaCl every other day; on day 5, 3mL of 1/4MS nutrient solution containing the test bacteria was added to each plastic cup (soil bacteria concentration was maintained at 10%⁸Per gram of soil).

4. After step 3 was completed, the plant height and dry fresh weight of corn and wheat were determined.

3 replicates were set for each strain tested.

A control (Salt control) without inoculated strain was set.

A control (Mock control) was set up using deionized water instead of deionized water containing 200mM NaCl, and without inoculating the strain.

The observation of the maize and wheat phenotypes of the inoculated strain T1-8 is shown in FIG. 3. In FIG. 3, A is corn and B is wheat.

Fourthly, morphological identification and molecular identification of strain T1-8

1. Bacterial colonies of the strain T1-8 on an LB solid medium are flat, irregular in edge, white and sticky, and have wrinkles on the surface.

2. The 16S rDNA sequence of the strain T1-8 is amplified and sequenced, and the sequencing result is shown as the sequence 1 in the sequence table.

Through identification, the strain T1-8 can be determined to belong to the bacillus licheniformis, and therefore, the strain is renamed to the bacillus licheniformis T1-8.

Fifth, preservation of Bacillus T1-8

The Bacillus licheniformis (Bacillus paracheniformis) T1-8 provided by the invention has been preserved in China general microbiological culture Collection center (CGMCC for short; No. 3 of Beijing university Hokko No.1 of Shangyang district, China academy of sciences microbial research, and zip code 100101) in 03 and 15 days in 2019, and the preservation number is CGMCC No. 17335. Bacillus paracasei (Bacillus paraceriniformis) T1-8 is simply called Bacillus T1-8.

Example 2 determination of the content of stress-related substances in maize plants inoculated with Bacillus T1-8

4. After completion of step 3, the water in the keg was replaced with 1/4MS nutrient solution containing 140mM NaCl, while the roots of the corn were inoculated with 10ml of 1/4MS nutrient solution containing Bacillus T1-8 (bacteria concentration in 1/4MS nutrient solution was 10)⁷one/mL), incubated at 25 ℃ for 3 days.

3 replicates were set for each strain tested.

A control (Salt control) without inoculated strain was set.

5. And (4) after the step 4 is completed, determining the content of various substances (soluble total sugar, hydrogen peroxide, MDA, superoxide dismutase, catalase and hydrogen peroxide) related to salt stress in the corn leaves.

Measuring the content of soluble total sugar by using an anthrone colorimetric method;

measuring the content of hydrogen peroxide by a xylenol orange method;

MDA, superoxide dismutase, catalase, hydrogen peroxide were detected using a commercially available kit (all purchased from solibao biotechnology limited).

The method for measuring the content of the soluble total sugar by the anthrone colorimetric method comprises the following steps:

making standard curve

Taking 6 test tubes with 20mL scales, numbering from 1 to 6 respectively, adding 100 mu g/mL sucrose solutions with 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL, supplementing the solutions with water to 2mL, wherein the sugar content in each test tube is 0, 20, 40, 60, 80 and 100 mu g respectively, then sequentially adding 0.5mL anthrone ethyl acetate reagent and 5mL concentrated sulfuric acid into the test tubes, fully oscillating, immediately placing the test tubes into a boiling water bath, accurately preserving the heat for 1min tube by tube, taking out, naturally cooling to room temperature, measuring the absorbance of the test tubes at a wavelength of 630nm by taking a blank as a reference, drawing a standard curve by taking the absorbance as a vertical coordinate and the sugar content as a horizontal coordinate, and solving a standard linear equation.

② extraction of soluble sugar

Weighing 0.10-0.30 g of crushed sample, putting the crushed sample into a 20mL graduated test tube, adding 5-10 mL of distilled water, sealing the test tube by using a plastic film, extracting the sample in boiling water for 30min (extracting for 2 times), filtering an extracting solution into a 25mL volumetric flask, repeatedly washing the test tube and residues, and fixing the volume to the graduation.

③ color development determination

Sucking 0.5mL of sample liquid into a 20mL graduated test tube (repeated 3 times), adding 1.5mL of distilled water, adding the anthrone ethyl acetate reagent and the concentrated sulfuric acid solution in sequence according to the steps of preparing a standard curve, developing and measuring the absorbance. The amount of sugar (. mu.g) was determined from a standard linear equation and the sugar content in the test sample was calculated.

The method for measuring the hydrogen peroxide content by the xylenol orange method comprises the following steps:

making standard curve

Taking six 10mL centrifuge tubes, adding 0, 0.4, 0.8, 1.2, 1.6 and 2.0mL 10 mu mol/L hydrogen peroxide respectively, adding water to 2mL, adding 4mL working reagent into each tube during color development, developing in water bath at 30 ℃ for 30min, measuring the absorbance value at 560nm, and drawing a standard curve.

Extraction of sample

Taking 0.5g of plant tissue sample, adding 2mL of precooled acetone, grinding and homogenizing, centrifuging at 10000r/min for 10min, fixing the volume of the supernatant to 3mL to obtain hydrogen peroxide extract, taking 1mL of the supernatant, adding 3mL of extracting agent, mixing uniformly, adding 5mL of distilled water, mixing uniformly, centrifuging at 5000r/min for 1min, and taking the upper-layer water phase as sample extract.

③ color development determination

Taking 2mL of sample extracting solution, adding 4mL of working reagent, processing according to a standard curve method, measuring an absorbance value at 560nm, and checking the concentration of hydrogen peroxide from the standard curve.

The results are shown in FIG. 4. The result shows that the inoculation of the bacillus T1-8 can improve the Total Soluble Sugar (TSS) and the Peroxidase (POD) activity in the corn leaves and simultaneously can reduce the Malondialdehyde (MDA) and the hydrogen peroxide (H) in the corn leaves compared with the control group₂O₂) Content, superoxide dismutase (SOD) and Catalase (CAT) activity.

Example 3 determination of the growth promoting ability of Bacillus T1-8

One, NH₃Productivity assay

Inoculating Bacillus T1-8 into a test tube containing peptone water culture solution at 28 deg.C and 170rmin^-1Shaking for 72h, adding 1mL Nessler's reagent (0.09mol/L mercuric iodide and 2.5mol L)^-1Potassium hydroxide mixture), the strain with yellow brown precipitate is positive reaction for producing ammonia.

Peptone water culture fluid: 10g of peptone powder, 5g of NaCl and distilled water are added until the volume is 1000mL and the pH value is 7.8.

Measurement of siderophore production ability

The bacillus T1-8 is spotted on an LB solid culture medium plate, the bacillus T1-8 is cultured for 24h at 28 ℃, then a CAS solid culture medium is poured and laid on the LB plate, after 15-30min, whether a yellowish or reddish halo appears around the bacteria is observed, and the siderophin producing capability of the bacteria is preliminarily judged.

Inoculating Bacillus T1-8 to LB liquid culture medium, culturing at 28 deg.C and 170rpm for 12h, collecting culture supernatant, sterilizing with membrane, mixing with CAS blue detection solution in equal volume, reacting for 30min, and measuring OD₆₃₀Obtaining a light absorption value As, and taking double distilled water As contrast for zero adjustment; the un-inoculated culture solution and the CAS blue detection solution have equal volumeMixing and measuring OD₆₃₀To obtain the light absorption value Ar. The As/Ar value represents the relative content of the siderophin in the sample, the siderophin content measurement is repeated for 3 times, and the lower the As/Ar value is, the stronger the siderophin production capacity is. Generally, the As/Ar of the strain with higher siderophore production capacity is less than 0.5.

Third, IAA Generation ability measurement

Inoculating Bacillus T1-8 to a culture medium containing 2.5mmol L^-1In Landy medium of L-tryptophan at 25 deg.C for 140r min^-1Shaking and culturing for 72h, collecting culture solution 1mL, centrifuging at 12000 Xg for 5min to collect supernatant, collecting supernatant 500 μ L, adding equal volume Salkowski reagent, developing color at room temperature in dark for 30min, measuring optical density value at 530nm, using blank culture medium as control, and using optical density corresponding to pure IAA as standard curve to calculate IAA output (mg L)^-1)。

Fourthly, determination of phosphate-solubilizing ability

Activating Bacillus T1-8 in LB culture medium, collecting 1mL culture solution, 6000r min^-1Centrifuging for 5min, removing supernatant, washing thallus with sterile water for three times, resuspending, inoculating Bacillus T1-8 into phosphate liquid Nutrient (NBRIP), inoculating bacteria as blank control, and culturing at 28 deg.C for 170r min^-1The shaking culture is carried out for 7 days under the condition, and the content of the dissolved phosphorus and the corresponding pH value are quantitatively measured by a molybdenum-antimony colorimetric-resistance method.

The results are shown in Table 1.

TABLE 1

The result shows that the bacillus T1-8 has the capability of producing ammonia, decomposing inorganic phosphorus, producing siderophin and the like, and is the rhizosphere growth promoting bacillus.

Example 4 hemolysis reaction of Bacillus T1-8

The bacillus T1-8 is inoculated on a sheep blood plate, inverted culture is carried out for 24h at 37 ℃, no hemolysis cycle is observed, namely the hemolysis reaction is negative, and the bacillus T1-8 can be used for developing and utilizing fertilizers.

Sheep blood plates: 10g of peptone, 10g of beef extract powder, 5g of sodium chloride, 15g of agar, and distilled water to a constant volume of 1000mL, pH7.5, sterilizing under high pressure (121 ℃, 15min), cooling to about 60 ℃, adding 60mL of sterile defibered sheep blood, fully shaking in a rotary manner, and then preparing a flat plate. The blood agar layer is 4mm thick.

Example 5 liquid fermentation of Bacillus T1-8

The bacillus T1-8 is inoculated in a fermentation medium for fermentation.

Solutes of a fermentation medium (pH 7.5 + -0.1) used for fermentation include a carbon source, a nitrogen source, sodium chloride, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, ferrous sulfate, zinc sulfate and manganese sulfate; the solute is water. Wherein the concentrations of part of the solutes and their fermentation medium are: 0.5g/100ml of sodium chloride, 0.1g/100ml of monopotassium phosphate, 0.1g/100ml of calcium chloride, 0.1g/100ml of magnesium sulfate, 0.05g/100ml of ferrous sulfate, 0.05g/100ml of zinc sulfate and 0.05g/100ml of manganese sulfate.

Designing a single-factor experiment as shown in Table 2 according to carbon source, nitrogen source, carbon-nitrogen ratio, fermentation temperature, liquid loading amount, inoculation amount, pH of fermentation system, and fermentation speed in liquid fermentation medium, and counting OD in fermentation liquid at the end of fermentation₆₀₀. The carbon content, nitrogen content and carbon-nitrogen ratio of each carbon source and nitrogen source in Table 2 are shown in Table 3.

The inoculum size in Table 2 is 10⁹The volume percentage of the LB liquid culture medium of the bacillus T1-8/mL when inoculated in a fermentation system is shown in brackets as the bacteria concentration in the initial fermentation system after inoculation.

TABLE 2

TABLE 3

The results are shown in FIG. 5. Highest OD in fermentation broth₆₀₀＝6.76。

From the results of FIG. 5, orthogonal experiments (24 h per set of settings) as shown in Table 4 were designedAnd 48h two fermentation times). The OD of the fermentation liquor at the end of the fermentation is counted₆₀₀Or the amount of spores.

TABLE 4

The results are shown in FIG. 6. The highest OD in the fermentation liquor after 24h fermentation₆₀₀12.11, highest OD in fermentation broth after 48h of fermentation₆₀₀12.98, the number of spores can reach 1 × 10¹⁰1.5X 10 per mL¹⁰one/mL.

According to the results, the solutes of the optimal fermentation medium (pH 7.5 +/-0.1) of the bacillus T1-8 are determined to be molasses, soybean meal powder, sodium chloride, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, ferrous sulfate, zinc sulfate and manganese sulfate; the solute is water. Wherein the solute and the concentration thereof in the fermentation medium are: 5g/100ml of molasses, 0.72g/100ml of soybean meal, 0.5g/100ml of sodium chloride, 0.1g/100ml of monopotassium phosphate, 0.1g/100ml of calcium chloride, 0.1g/100ml of magnesium sulfate, 0.05g/100ml of ferrous sulfate, 0.05g/100ml of zinc sulfate and 0.05g/100ml of manganese sulfate.

The bacillus T1-8 was inoculated into the fermentation medium at 5% inoculum size and cultured at 35 ℃ and 180rpm for 48 hours. Under the condition, the amount of viable bacteria or spores in the fermentation liquor is more than or equal to 1 multiplied by 10 when the fermentation is finished¹⁰one/mL.

Example 6 preparation of microbial organic fertilizer

The organic materials for preparing the microbial organic fertilizer are purchased from Unionvered Biotech limited of Jiangyun City, and are decomposed cow dung, pig dung and straws mixed compost, wherein the total nitrogen content is 1.56%, the total phosphorus content is 2.52%, the total potassium content is 1.32%, the organic matter content is 48.51%, the water content is 28.26%, and the pH value is 6.8. Before the secondary fermentation, the organic materials are supplemented to 50 to 65 percent by clear water and then the secondary fermentation is carried out.

1. Bacillus T1-8 was inoculated into the optimal fermentation medium selected in example 5 at an inoculum size of 5%, and cultured at 35 ℃ and 180rpm for 48 hours to obtain a liquid fermented broth (in this case, the concentration of the broth was about 1X 10¹⁰-1.5×10¹⁰one/mL).

2. Uniformly mixing the liquid zymophyte liquid obtained in the step (1) with organic materials, performing secondary solid fermentation in a ventilated and cool place, and adding 30L of the liquid zymophyte liquid into each ton of the organic materials (namely adding 3 multiplied by 10 to each ton of the organic materials)¹⁴-5×10¹⁴And (3) performing one-time pile turning on the bacillus T1-8) in the fourth day, keeping the fermentation temperature of the bacillus T1-8 at 30-50 ℃, controlling the water content of a fermentation system at 50-60%, and finishing the fermentation after 7 days to obtain the microbial organic fertilizer.

Through detection, the amount of viable bacteria or spores in the microbial organic fertilizer reaches 5 multiplied by 10⁸-2×10⁹Per gram.

Example 7 verification of fertilizer efficiency of microbial organic fertilizer by field plot experiment

Selecting three saline-alkali soil with different saline-alkali degrees (3 per thousand salt land blocks, 4 per thousand salt land blocks and 5 per thousand salt land blocks) in a Bohai sea granary without a juneberry demonstration sample plate project area from late 6 months to early 10 months in 2018, performing plot test on the three saline-alkali soil with a variety of a constant source corn I, respectively applying different fertilizer treatments (the treatment method is shown in the table 5), sampling and measuring the yield of the corn after three months, simultaneously taking a soil sample to measure the physical and chemical properties of the soil (the soil organic matter content is measured by a potassium dichromate oxidation-external heating method, the soil total nitrogen content is measured by a semi-micro Kjeldahl method, the soil total phosphorus content is measured by a NaOH alkali fusion molybdenum antimony anti-spectrophotometry method, the soil total salt content is measured by a residue drying-weight method, the soil pH is measured by a glass electrode method, the soil cation exchange amount is measured by a sodium acetate extraction-atomic spectrophotometry method, the soil chloride ion content is measured by a silver nitrate titration method, the soil total phosphorus content is measured by a NaOH alkali fusion molybdenum antimony nitrate alkali fusion Digesting by hydrogen peroxide-hydrofluoric acid, and measuring the content of potassium and sodium elements in the soil by ICP-AES; the method is described in a document 'Liangyuyan, nitric acid-hydrofluoric acid-hydrogen peroxide system digestion of heavy metal [ J ] in soil, Guangdong chemical industry, 2017 (8)') to verify the fertilizer efficiency of the microbial organic fertilizer.

In Table 5, the compound fertilizer is purchased from fertilizer seed agricultural pharmacy in Amelanchier county, wherein N-P2O5-K2O is 16-16-16; the common organic fertilizer is an organic material used for preparing the microbial organic fertilizer in example 5, is purchased from the integrated biotechnology limited of Jiangyun city, and is a decomposed mixed compost of cow dung, pig dung and straws, wherein the total nitrogen content is 1.56%, the total phosphorus content is 2.52%, the total potassium content is 1.32%, the organic matter content is 48.51%, the water content is 28.26, and the pH value is 6.8.

TABLE 5

The cell tests were each set to 3 replicates, randomly distributed. And (4) applying fertilizer to each cell, carrying out rotary tillage, sowing seeds after the rotary tillage, and watering every other day. Applying additional fertilizer once in the maize staminate extraction period. During top dressing, the plant height, stem thickness, dry fresh weight and the like of the corn are measured by sampling. And after three months, the corn is sampled to measure yield, and a soil sample is taken to measure the physical and chemical properties of the soil.

The appearance of the corn is shown in fig. 7. In FIG. 7, T1 corresponds to the result of no fertilizer application (CK) in Table 5, T2 corresponds to the result of conventional fertilizer application (NPK) in Table 5, T3 corresponds to the result of general organic fertilizer application (NPK (BIO)) in Table 5, and T4 corresponds to the result of T1-8 organic fertilizer application (T1-8(BIO)) in Table 5.

The corn yield statistics are shown in table 6. In table 6, a is the detection result of 3 ‰ salt mass; b is a detection result of 4 per mill of salt land; c is the detection result of 5 per mill salt land.

The results of the soil physical and chemical property measurements are shown in Table 7. As shown. In Table 7, a is the detection result of 3 ‰ salt-containing plots; b is a detection result of 4 per mill of salt land; c is the detection result of 5 per mill salt land.

In the above tables, T1 corresponds to the result of no fertilizer application (CK) in Table 5, T2 corresponds to the result of conventional fertilizer application (NPK) in Table 5, T3 corresponds to the result of ordinary organic fertilizer application (NPK (BIO)) in Table 5, and T4 corresponds to the result of T1-8 organic fertilizer application (T1-8(BIO)) in Table 5.

TABLE 6

TABLE 7

The results show that after the microbial organic fertilizer of the strain T1-8 is applied to the soil with different saline-alkali degrees, the organic matter content of the soil is increased, and the pH value and the salt content are reduced to some extent compared with the common fertilizer; the strain T1-8 microbial organic fertilizer can improve the physicochemical property of soil and the yield of crops, can relieve the damage of salt stress to the crops to a great extent, and can improve the yield of saline-alkali soil crops. The microbial organic fertilizer prepared by the invention can be specially used for increasing the yield of crops in saline-alkali soil.

Sequence listing

<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences

Nanjing university of agriculture

<120> rhizosphere growth-promoting bacterium for enhancing salt tolerance of crops, microbial fertilizer and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1463

<212> DNA

<213> Bacillus licheniformis (Bacillus paralicheniformis)

<400> 1

accatcggta actctatact gcgagctggc tccaaaggtt acctcaccga cttcgggtgt 60

tacaaactct cgtggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg 120

catgctgatc cgcgattact agcgattcca gcttcacgca gtcgagttgc agactgcgat 180

ccgaactgag aacagatttg tgggattggc ttagcctcgc ggcttcgctg ccctttgttc 240

tgcccattgt agcacgtgtg tagcccaggt cataaggggc atgatgattt gacgtcatcc 300

ccaccttcct ccggtttgtc accggcagtc accttagagt gcccaactga atgctggcaa 360

ctaagatcaa gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga 420

cgacaaccat gcaccacctg tcactctgcc cccgaagggg aagccctatc tctagggttg 480

tcagaggatg tcaagacctg gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc 540

caccgcttgt gcgggccccc gtcaattcct ttgagtttca gtcttgcgac cgtactcccc 600

aggcggagtg cttaatgcgt ttgctgcagc actaaagggc ggaaaccctc taacacttag 660

cactcatcgt ttacggcgtg gactaccagg gtatctaatc ctgttcgctc cccacgcttt 720

cgcgcctcag cgtcagttac agaccagaga gtcgccttcg ccactggtgt tcctccacat 780

ctctacgcat ttcaccgcta cacgtggaat tccactctcc tcttctgcac tcaagttccc 840

cagtttccaa tgaccctccc cggttgagcc gggggctttc acatcagact taagaaaccg 900

cctgcgcgcg ctttacgccc aataattccg gacaacgctt gccacctacg tattaccgcg 960

gctgctggca cgtagttagc cgtggctttc tggttaggta ccgtcaaggt gccgccctat 1020

tcgaacggta cttgttcttc cctaacaaca gagttttacg atccgaaaac cttcatcact 1080

cacgcggcgt tgctccgtca gactttcgtc cattgcggaa gattccctac tgctgcctcc 1140

cgtaggagtc tgggccgtgt ctcagtccca gtgtggccga tcaccctctc aggtcggcta 1200

cgcatcgtcg ccttggtgag ccgttacctc cccaactagc taatgcgccg cgggtccatc 1260

tgtaagtggt agctaaaagc caccttttat gattgaacca tgcggttcaa tcaagcatcc 1320

ggtattagcc ccggtttccc ggagttatcc cagtcttaca ggcaggttac ccacgtgtta 1380

ctcacccgtc cgccgctgac ctaagggagc aagctcccgt cggtccgctc gactgcatca 1440

caaatctgta agtagggttg atg 1463

Claims

1. Bacillus licheniformis (Bacillus paraleniformis) T1-8 with the preservation number of CGMCC No. 17335.

2. A fermentation product of Bacillus paraclicheniformis (Bacillus paraleniformis) T1-8 according to claim 1, characterized in that: the fermentation product is obtained by inoculating the Bacillus licheniformis (Bacillus paracasei) T1-8 in the claim 1 into a liquid fermentation culture medium for fermentation culture;

3. The use of Bacillus paracasei (Bacillus paraceriniformis) T1-8 according to claim 1 or the fermentation product according to claim 2 as follows (a1) or (a 2):

(a1) relieving the damage of salt stress to plants;

(a2) improving the salt stress resistance of the plants.

4. Use of Bacillus paracasei (Bacillus paraccheniformis) T1-8 according to claim 1 or the fermentation product according to claim 2 for the preparation of a microbial fertilizer.

5. A microorganismThe fertilizer contains 5 x 10 of microbial fertilizer per gram⁸-2×10⁹Bacillus paracasei (Bacillus paraceriniformis) T1-8 according to claim 1.

6. The microbial fertilizer according to claim 5, wherein: the microbial fertilizer meets the following conditions: (1) the mass percentage of the organic matter is more than 45%; (2) the sum of the mass percentage of the nitrogen, the phosphorus and the potassium is more than 5 percent; (3) the water content is 50-60%; (4) the pH value is 6.5 to 7.5.

7. A preparation method of the microbial fertilizer comprises the following steps: mixing a fermentation product of Bacillus paracasei (Bacillus paracerini) T1-8 according to claim 2 with an organic material to perform secondary solid fermentation, thereby obtaining the microbial fertilizer.

8. The microbial fertilizer according to claim 7, wherein:

the organic material meets the following conditions: (1) the mass percentage of the organic matter is more than 45%; (2) the sum of the mass percentage of the nitrogen, the phosphorus and the potassium is more than 5 percent; (3) the pH value is 6.5 to 7.5.

And/or the presence of a gas in the gas,

9. Use of the microbial fertilizer of any one of claims 5 to 8 as at least one of (b1) - (b 5):

(b1) relieving the damage of salt stress to plants;

(b2) improving the salt stress resistance of plants;

(b3) as a fertilizer for plants in saline and alkaline land;

(b4) the plant yield of the saline-alkali soil is improved;

(b5) the physicochemical property of the saline-alkali soil is improved.