CN117757659A

CN117757659A - Bacillus pumilus and application thereof in improving abiotic stress resistance of crops

Info

Publication number: CN117757659A
Application number: CN202311536170.7A
Authority: CN
Inventors: 王晓辉; 徐广�; 伍淼; 刘华梅; 胡虓; 倪浩翔; 霍瑞; 周华众; 彭一凡; 周玄; 李萍萍; 熊海燕
Original assignee: Wuhan Kernel Bio Tech Co ltd
Current assignee: Wuhan Kernel Bio Tech Co ltd
Priority date: 2023-11-17
Filing date: 2023-11-17
Publication date: 2024-03-26

Abstract

Bacillus pumilus and application thereof in improving abiotic stress resistance of crops, and belongs to the technical field of agricultural microorganisms. In order to screen out microorganisms which have better abiotic stress resistance and can better survive and colonize, 1 strain KN-601 which has better salt tolerance and wide growth pH range is separated from soil collected in severe saline-alkali soil, and the strain is determined to be bacillus pumilus by combining morphological characteristics and molecular biological identification results of the strain. The strain can also show better resistance to low-temperature stress and oligotrophic, can well colonize soil, contains plant hormones such as IAA and the like in fermentation liquor, can promote seedling growth, and improves seed emergence rate under saline-alkali stress. In addition, the microbial preparation prepared from the strain can improve the yield of crops such as rice under saline-alkali stress, and has good application prospect.

Description

Bacillus pumilus and application thereof in improving abiotic stress resistance of crops

Technical Field

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to bacillus pumilus and application thereof in improving the abiotic stress resistance of crops.

Background

Stress refers to the sum of various environmental factors that are detrimental to plant growth and survival, also known as stress. The plant is in a stress environment, so that the growth and development of the plant are seriously affected, and the biomass and yield of the plant are limited. There are many kinds of stress, but the stress can cause dehydration of cells, rupture of biological membranes, and disorder of various metabolism, and can be classified into biological stress and non-biological stress. Biological stress refers to the phenomenon that the disease, insect and weed damage causes, and abiotic stress is more in variety, and common in production such as high temperature, low temperature, drought, waterlogging, saline-alkali stress and the like. Stress causes plants to undergo a range of responses from affecting stress gene expression to cellular metabolism to growth and development. Some plants cannot adapt to these adverse environments and cannot survive. Studies have shown that crop yield losses caused by abiotic stress (e.g., low temperature, high salt, drought, etc.) and biotic stress (e.g., disease, pest, weed damage, etc.) are quite dramatic worldwide, with an average yield drop of 65% -85%.

Agricultural production faces double hazards of biotic stress and abiotic stress, the biotic stress can be prevented and intervened in time through artificial measures, but the abiotic stress is generally not manually controllable, once the damage area is wide, and the damage degree is high. It is counted that 60% -80% of the yield loss of crops is caused by abiotic stress, and huge economic loss is brought to human beings. In recent years, extreme weather is frequently appeared, adverse effects of abiotic stress on global agricultural production are aggravated, and abiotic stress has become an important factor for limiting crop yield improvement. Meanwhile, the frequency, degree and duration of the occurrence of the abiotic stress are obviously increased along with the global climate change, so that the resistance of crops to the abiotic stress is improved, or adverse effects of the abiotic stress on the crop yield and quality formation are reduced by taking measures, and the method has important significance for ensuring stable crop yield and grain safety.

When abiotic stress occurs, the plant may resist the stress by a physiological reaction, however if the stress effect exceeds the plant's own tolerance, beyond its ability to repair, the damage suffered by the plant will become irreversible and the plant will thus suffer and even die. The existing effective ways for reducing the damage of abiotic stress to crops are as follows: and cultivating strong seedlings and the like, and spraying and inducing the crops to generate stress-resistant biological stimulation and other combined comprehensive prevention and control measures, so as to improve the autoimmunity of the crops and resist the harm of external bad factors. Researches show that the microbial agent can promote the growth of crops, can induce plants to synthesize more substances for improving the stress resistance of the plants, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), proline and the like, and is a widely applied method in the current agricultural production. However, the quality of microbial agents in the current market is uneven, and the product use effect is possibly unstable due to the different survival and colonization capacities of strains in the product in different environments. Thus, there is a need to screen microorganisms that have better resistance to abiotic stress and are able to better survive colonization for increasing the resistance of plants under abiotic stress.

Disclosure of Invention

In order to screen out microorganisms which have good abiotic stress resistance and can better survive and colonize, the invention screens out a strain of plant growth hormone, has good abiotic stress resistance and can better survive and colonize Bacillus pumilus, and the strain is used for resisting the abiotic stress of plants so as to promote the growth of crops and further improve the yield of the crops.

In order to solve the technical problems and realize the corresponding technical effects, the invention provides the following technical scheme:

the first object of the invention is to provide a bacillus pumilus KN-601, wherein the bacillus pumilus KN-601 is preserved in China Center for Type Culture Collection (CCTCC) NO: m20221316, the preservation date is 2022, 8 and 24 days, and the preservation unit address is China, wuhan and university of Wuhan.

The second object of the invention is to provide the application of the bacillus pumilus KN-601 in improving the abiotic stress resistance of crops.

In one embodiment of the invention, the abiotic stress is a low temperature stress, a salt-alkali stress, an acid-base stress or an oligotrophic stress.

The third object of the invention is to provide an application of the bacillus pumilus KN-601 in improving the seed emergence rate under the saline-alkali stress.

The fourth object of the invention is to provide an application of the bacillus pumilus KN-601 in promoting crop growth and improving crop yield under non-stress conditions.

The fifth object of the present invention is to provide a microbial agent containing the above Bacillus pumilus KN-601.

In one embodiment of the invention, the microbial agent is a single-agent product of bacillus pumilus KN-601 or a compound product prepared by bacillus pumilus KN-601 and any one or more than two of bacillus, paenibacillus and fungus.

In one embodiment of the invention, the compound product is a microbial agent prepared from bacillus pumilus KN-601, bacillus amyloliquefaciens and bacillus licheniformis.

In one embodiment of the invention, the compound product is a microbial agent prepared from bacillus pumilus KN-601 and Paenibacillus polymyxa.

In one embodiment of the invention, the compound product is a microbial agent prepared from bacillus pumilus KN-601 and trichoderma subtilis.

In one embodiment of the invention, the microbial agent is in a dosage form including, but not limited to, liquid, granule, powder.

In one embodiment of the invention, the effective viable count in the microbial agent is not less than 1×10 ⁹ CFU/g。

The sixth object of the invention is to provide a biological bacterial fertilizer containing the bacillus pumilus KN-601.

The seventh object of the invention is to provide the application of the microbial agent or the biological bacterial fertilizer in improving the abiotic stress resistance of crops, promoting plant growth and improving crop yield.

The invention has the beneficial effects that:

1. the bacillus pumilus KN-601 screened by the method has the characteristics of salt and alkali resistance, low temperature resistance, wide growth pH range, and proliferation under oligotrophic conditions.

2. Bacillus pumilus KN-601 can produce plant growth hormone and amino acid in high yield during proliferation process, and can promote crop growth.

3. The fermentation liquor of the bacillus pumilus KN-601 can improve the emergence rate of seeds under the condition of saline-alkali stress.

4. Bacillus pumilus KN-601 can still survive and proliferate well in saline-alkali soil.

5. The microbial inoculum product containing the bacillus pumilus KN-601 can improve the yield of crops such as paddy rice, wheat, potato and the like in saline-alkali soil, and the yield can be increased by more than 25 percent; in addition, the microbial inoculum product containing the bacillus pumilus KN-601 can also improve the tolerance of wheat to low temperature (cold in the spring) and prevent the wheat from being frozen.

6. The microbial inoculum product containing the bacillus pumilus KN-601 can also improve the yield of cowpea under non-saline-alkali stress.

Drawings

FIG. 1 is a graph showing the growth of 5 strains of bacteria obtained by the preliminary screening in example 1 on a Gibben modified medium plate containing 8-12% salt;

FIG. 2 is a morphology of a single colony of Bacillus pumilus KN-601 on NA medium;

FIG. 3 is a phylogenetic tree diagram of Bacillus pumilus KN-601;

FIG. 4 is a graph showing the growth of Bacillus pumilus KN-601 at different temperatures, A in FIG. 4 is a graph showing the growth of Bacillus pumilus KN-601 at different temperatures, and B in FIG. 4 is a graph showing the growth of Bacillus pumilus ACCC 01891 at different temperatures;

FIG. 5 is a graph showing the growth of Bacillus pumilus KN-601 on LB solid medium plates diluted 10-fold, wherein the control in FIG. 5 is a graph showing the growth of Bacillus pumilus ACCC 01891 on LB solid medium plates diluted 10-fold;

FIG. 6 is a graph showing the growth promoting effect of Bacillus pumilus KN-601 fermentation broth on maize and rice seedlings; wherein A in FIG. 6 is a graph of the growth promoting effect of the fermentation broth of Bacillus pumilus KN-601 on corn seedlings, and B in FIG. 6 is a graph of the growth promoting effect of the fermentation broth of Bacillus pumilus KN-601 on rice seedlings;

FIG. 7 is a graph showing the effect of Bacillus pumilus KN-601 fermentation broth on wheat germination rate and rice seedling emergence rate under saline-alkali stress conditions; wherein A in FIG. 7 is a graph of the effect of the fermentation broth of Bacillus pumilus KN-601 on the germination rate of wheat under the condition of salt-alkali stress, and B in FIG. 7 is a graph of the effect of the fermentation broth of Bacillus pumilus KN-601 on the germination rate of rice under the condition of salt-alkali stress;

FIG. 8 is a graph showing the effect of the compound microbial agent on the rice seedling emergence rate in saline-alkali soil in example 10;

FIG. 9 is a graph showing the effect of the composite microbial preparation on the growth state of wheat seedlings at low temperature in example 11; wherein, A in figure 9 is a wheat seedling growth state diagram of a peasant household conventional management area, and B in figure 9 is a wheat seedling growth state diagram of a compound microorganism preparation treatment area;

FIG. 10 is a graph showing the effect of the compound microorganism on cowpea growth under non-saline-alkali stress in example 13; wherein, a in fig. 10 is a graph of the growth of cowpea in a peasant household conventional management area, and B in fig. 10 is a graph of the growth of cowpea in a compound microbial agent treatment area.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the following detailed description and the accompanying drawings. The following specific examples are provided to further illustrate the technical aspects of the present invention, but the scope of the present invention is not limited to these examples. All changes and equivalents that do not depart from the gist of the invention are intended to be within the scope of the invention.

The experimental procedures used in the examples below were conventional, and the materials, reagents and apparatus used, unless otherwise indicated, were conventional in the art and are commercially available to those skilled in the art.

Bacillus pumilus ACCC 01171, bacillus pumilus ACCC 01891 and Bacillus pumilus ACCC 01940 used in the invention are purchased from China center for type culture Collection of microorganisms.

Paenibacillus polymyxa (BaciIllus polymyxa) KN-03 used in the present invention is disclosed in patent document with the application number of CN201410777999.0, and the name of the invention is Bacillus polymyxa KN-03, and a culture method and application thereof.

Trichoderma citrinoviride (Trichoderma citrinoviride) KN-T108 used in the invention is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m20221540, the preservation date is 2022, 9 and 30 days, and the preservation unit address is China, wuhan and university of Wuhan.

Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KN-530 used in the invention is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m20221315, the preservation date is 2022, 8 and 24 days, and the preservation unit address is China, wuhan and university of Wuhan.

The bacillus licheniformis (Bacillus licheniformis) KN-403 used in the invention is preserved in China Center for Type Culture Collection (CCTCC) NO: m20221317, the preservation date is 2022, 8 and 24 days, and the preservation unit address is China, wuhan and university of Wuhan.

The Gibben modified culture medium containing 5% -12% of salt comprises the following components: 10.0g of peptone, 5.0g of beef extract, 50 g-120 g of NaCl, 30g of agar and 1000mL of distilled water, and the pH value is 7.0-7.2.

Activating bacillus pumilus KN-601 strain: selecting a loop of bacillus pumilus KN-601, inoculating the loop of bacillus pumilus KN-601 into a liquid culture medium, and placing the inoculated liquid shake flask into a reciprocating constant temperature shaking table at 30 ℃ for culture at a rotating speed of 160rpm; and (3) picking a loop of the cultured bacterial suspension, inoculating the loop to a slant culture medium, and placing the inoculated slant in a 30 ℃ water-proof constant-temperature incubator for culturing for 72 hours to obtain the activated bacillus pumilus KN-601.

The composition of the liquid medium (liquid LB) was: 10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride and 1000mL of tap water, wherein the pH value is 7.0-7.2, and sterilizing for 30min at 121 ℃ after uniform mixing.

The composition of the slant culture medium is as follows: 10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride, 15g of agar powder and 1000mL of tap water, wherein the pH value is 7.0-7.2, and sterilizing for 30min at 121 ℃ after uniform mixing.

The seed culture method of the bacillus pumilus KN-601 comprises the following steps: inoculating activated Bacillus pumilus into seed culture medium with an inoculum size of 1%, and culturing in a reciprocating constant temperature shaker at 30deg.C and 160rpm for 7-9 hr.

The composition of the seed culture medium is as follows: 0.8% of soybean meal powder, 1% of D-xylose, 0.5% of yeast powder, 1% of corn steep liquor, 0.1% of monopotassium phosphate and 6.5-7.2 of pH value.

The fermentation medium for preparing the bacillus pumilus KN-601 fermentation broth comprises the following components: 2% of soybean meal powder, 3% of D-xylose, 0.5% of yeast powder, 1.2% of corn steep liquor, 1.5% of corn starch, 0.1% of monopotassium phosphate, 0.03% of calcium chloride, 0.15% of magnesium sulfate, 0.02% of manganese chloride and the balance of water, wherein the pH value is 7-7.2.

The method for preparing the bacillus pumilus KN-601 fermentation liquor comprises the following steps: and (3) inoculating the prepared bacillus pumilus KN-601 seed solution into a fermentation medium, wherein the early-stage culture temperature is 30 ℃, adding glycine with the final concentration of 0.5% (mass concentration) into the fermentation medium when the bacillus pumilus KN-601 seed solution is cultured to the logarithmic phase, continuously culturing until more than 90% of nutrients form spores, controlling the culture temperature at 28 ℃, controlling the dissolved oxygen at 30%, and continuously culturing for 5 hours to obtain the bacillus pumilus KN-601 fermentation solution.

Example 1: isolation, screening and identification of strains

(1) Separation and screening of Bacillus pumilus KN-601

And (3) collecting a soil sample from Ningxia Pingrow severe saline-alkali soil, separating microorganisms, weighing 10g of saline-alkali soil sample, adding 90mL of sterile water, placing in a shaking table 180r/min for shaking for 20min, diluting the soil leaching solution by adopting a 10-fold dilution method, sucking 100 microliters of diluted soil leaching solution by using a pipette, uniformly coating the diluted soil leaching solution on a Gibben modified culture medium flat plate containing 5% of salt, and inversely culturing for 2-3d in a constant-temperature incubator at 30 ℃. Colonies with different forms grow on the plate of the Gibben modified culture medium after the culture, single colonies of the different colonies are purified by a plate streaking method, and marked and placed in an incubator for 2-3d culture.

The 5 strains obtained by the primary screening were streaked and re-screened on Gibben modified medium plates containing 8-12% of salt, and the re-screening results are shown in FIG. 1. From FIG. 1, it is clear that strains KN-601 and KN-403 can grow well on Gibben modified medium plates containing 8% and 10% of salt, and strain KN-601 can also grow on Gibben modified medium plates containing 12% of salt.

Shake flask culture was performed on 5 strains obtained by re-screening in liquid LB medium with pH of 4.0-10.0, and proliferation of each strain after 10h shake culture was measured, and the results are shown in Table 1. As is clear from Table 1, the strain KN-601 was still able to be increased in the LB medium having pH of 4.0 and pH of 10.0, compared with the other four strains, indicating that Bacillus pumilus KN-601 was suitable for a wide range of growth and a large number of soil types.

TABLE 1 OD after shaking culture of the strains in LB medium of different pH values for 10h ₆₀₀ Growth rate

Based on the alkaline soil, the soil is alkaline, and the salt content in the soil is higher, so that the screened strains KN-601, KN-403 and KN-611 are further re-screened under the condition of salt and alkali combination, and the growth condition of the strains under the environment of high salt and high pH is determined. The 3 strains further subjected to the rescreening were subjected to shaking culture in liquid medium (tryptone 10g, yeast extract 5g, sodium chloride 100g, tap water 1000 mL) at pH 7.0-10.0, and proliferation of each strain after shaking culture for 10 hours was measured, and the results are shown in Table 2. As shown in Table 2, the strain KN-601 was still able to be increased in a high-salt and high-alkaline medium, compared with the other two strains. Therefore, the strain KN-601 has good saline-alkali resistance and wide growth pH range, so that the strain KN-601 is identified.

TABLE 2 OD after shaking culture of the strains in high-salt LB medium of different pH values for 10h ₆₀₀ Growth rate

(2) Identification of Bacillus pumilus KN-601

(1) Morphological identification

The strain KN-601 obtained by re-screening is inoculated on nutrient agar (beef extract peptone) flat plate culture medium, the culture is carried out at the constant temperature of 30 ℃, the colony morphology is observed after a single colony grows, the thallus size, morphology, edge and transparency are recorded, and a small amount of thallus is coated on a glass slide by an inoculating loop for gram staining and microscopic examination. The colony morphology of the strain KN-601 is shown in FIG. 2, and as can be seen from FIG. 2, the colony of the strain KN-601 on NA medium is nearly circular, off-white and opaque. The bacterial strain KN-601 is observed by an optical microscope to find that the bacterial strain KN-601 is in a rod shape, round end and single or short chain arrangement, and the bacterial strain size is (0.6-0.7) mu m multiplied by (2.0-3.0) mu m; the spore ellipse is located in the center of the thallus or slightly deviated, the thallus is not expanded after the spore is formed, the membranous inclusion is avoided, the periphyton can move, and the strain KN-601 is found to be gram positive bacteria through gram staining.

(2) Physiological and biochemical identification

The invention also determines the physiological and biochemical characteristics of the bacillus pumilus KN-601, namely the enzyme activity and the carbon source utilization condition, and the specific results are shown in tables 3 and 4.

TABLE 3 physiological and biochemical characteristics of strain KN-601-carbon source utilization

Note that: positive reaction; negative reaction; w, weak positive reaction

TABLE 4 physiological and biochemical characteristics of strain KN-601-enzyme Activity

Note that: positive reaction; negative reaction; w, weak positive reaction

(3) Molecular biological identification

The flat plate of the strain KN-601 is sent to China center for sequencing and identification of 16S rRNA, and the sequencing result is shown as SEQ ID NO.1.

SEQ ID NO.1：

ATGCAAGTCGAGCGGACAGAAGGGAGCTTGCTCCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCGAGAGTAACTGCTCGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACCGCAAGGTTTAGCCAATCCCATAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGG

After completion of the sequencing, the determined sequence of the KN-60116S rRNA gene was subjected to BLAST alignment in NCBI database, and the 16S rRNA sequence was found to be most similar to Bacillus pumilus ATCC 7061. And selecting a standard strain which has relatively close relationship with the strain KN-601, performing phylogenetic analysis by adopting MEGA7 software, and constructing a phylogenetic tree, as shown in figure 3. As is clear from FIG. 3, the strain KN-601 is on the same branch as Bacillus pumilus and has stable relationship. Thus, the strain KN-601 was identified as Bacillus pumilus (Bacillus pumilus) in combination with morphological characteristics, physiological and biochemical characteristics, enzyme activities, and carbon source utilization of the strain KN-601.

The strain KN-601 is preserved in China Center for Type Culture Collection (CCTCC) NO: m20221316, the preservation date is 2022, 8 and 24 days, and the preservation unit address is China, wuhan and university of Wuhan.

Example 2: functional study of Bacillus pumilus KN-601

In order to further study the functional characteristics of the bacillus pumilus KN-601, the invention tests the low temperature resistance and proliferation capacity of the bacillus pumilus KN-601 under oligotrophic conditions.

Low temperature resistance test: the Bacillus pumilus KN-601 was streaked onto beef extract peptone plates, and cultured in the environments of 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃ and 30 ℃ respectively, with 28 ℃ as a Control (CK), 3 plates were used for each temperature gradient, and after 5 days, the growth of KN-601 on each plate was investigated (see FIG. 4), and the control was inoculated with Bacillus pumilus ACCC 01891. As can be seen from FIG. 4, bacillus pumilus KN-601 was able to grow on beef extract peptone plates at 5 ℃.

Oligotrophic assay: bacillus pumilus KN-601 was streaked onto plates of 10-fold diluted LB medium, control inoculated with Bacillus pumilus ACCC 01891, left to stand at 30℃for cultivation, and after 3 days the growth of KN-601 on the plates was investigated (see FIG. 5). As can be seen from FIG. 5, bacillus pumilus KN-601 still grew normally on oligotrophic plates.

Example 3: application of bacillus pumilus KN-601 in promoting plant growth

(1) Bacillus pumilus KN-601 fermentation liquor for promoting plant seedling growth

The Bacillus pumilus KN-601 fermentation broth was diluted 100 times, the corn and rice which have just emerged were root-irrigated, seedlings which were not root-irrigated with the Bacillus pumilus KN-601 fermentation broth were used as a control, the plant height and root length were investigated 7 days after the treatment, and the fresh weights of the above-ground and underground parts were measured, and 30 plants were randomly investigated for each treatment, and specific results are shown in Table 5 and FIG. 6. As is clear from Table 5 and FIG. 6, root irrigation of corn and rice seedlings with Bacillus pumilus KN-601 fermentation broth can increase the plant height of corn and rice seedlings and the fresh weight of the aerial parts, i.e., bacillus pumilus KN-601 can promote plant seedling growth.

Table 5KN-601 fermentation liquor root irrigation treatment effect questionnaire on maize and rice seedling growth

(2) Production of plant hormone and amino acid by bacillus pumilus KN-601 fermentation liquor

And (3) taking a bacillus pumilus KN-601 fermentation broth during fermentation tank placement, immediately placing the fermentation broth in a refrigerator at the temperature of-70 ℃ for freezing preservation, and sending a sample to the WU-Pu Nesi biotechnology Co., ltd, and detecting phytohormone and amino acid in the fermentation broth, wherein the detection result is shown in Table 6.

The Bacillus pumilus ACCC 01891 is fermented by adopting the same fermentation medium and the same culture condition as those of the Bacillus pumilus KN-601, and the sample treatment method after the fermentation is finished is the same as that of the KN-601.

TABLE 6 Table 6 partial phytohormone and amino acid contents in Bacillus pumilus KN-601 fermentation broth

As shown by the detection results of Table 6, the Bacillus pumilus KN-601 produces high yield of plant hormone and amino acid in the proliferation process, for example, the content of auxin IAA in a fermentation broth is 420.838ng/mL, which proves that KN-601 can promote growth, and can also improve the tolerance of crops to saline-alkali stress (IAA can indirectly regulate the content of antioxidant enzyme activity and proline in plants, and IAA strains can stimulate the synthesis of endogenous IAA in plants, and can indirectly stimulate the content of proline and antioxidant enzyme activity in plants to offset ROS caused by salt stress, and simultaneously reduce the release amount of ethylene and the content of malondialdehyde); meanwhile, the fermentation liquor of the bacillus pumilus KN-601 is also rich in various amino acids, for example, the content of proline reaches 20.44ug/mL, and the proline can keep osmotic pressure in a plant body, so that the plant does not lose water under salt stress.

Example 4: application of bacillus pumilus KN-601 in promoting seed emergence under saline-alkali stress

Plate germination test: spreading 2 layers of water-absorbing filter paper on a 9 cm disposable culture dish, adding 15mL of saline alkali water (prepared by sodium chloride, sodium sulfate, sodium bicarbonate and sodium carbonate according to a molar volume ratio of 1:9:9:1) into the culture dish, and diluting wheat seeds (the Bacillus pumilus KN-601 fermentation liquor is diluted to a spore number of 1 multiplied by 10) ⁶ CFU/mL, soaking wheat seeds in short KN-601 diluent for 30 min), placing in culture dishes, and placing 30 seeds in each dish; then, the wheat seeds were cultured in an incubator at 25℃for 5 days, and the germination rates of the wheat seeds were examined, and the wheat seeds were immersed in dilutions of the fermentation broths of Bacillus pumilus ACCC 01171 and Bacillus pumilus ACCC 01891, respectively, in other treatment groups, and the examination results are shown in Table 7 and A in FIG. 7.

Potting test: filling a seedling culture nutrition substrate into a nutrition pot, pouring saline alkali water (prepared by sodium chloride, sodium sulfate, sodium bicarbonate and sodium carbonate according to the molar volume ratio of 1:9:9:1) accounting for 40% of the mass of the seedling culture substrate, then sowing rice seeds, covering about 1cm of the substrate after sowing every 30 seeds in the nutrition pot, and finally pouring a diluent of a bacillus pumilus KN-601 fermentation broth in every pot, wherein the fermentation broth is diluted to a spore number of 1 multiplied by 10 ⁶ CFU/mL, 30mL of diluent was poured per bowl, 3 replicates per treatment. And (5) surveying the emergence rate after the rice seedlings emerge, and calculating the average emergence rate. The other treatment groups were watered with dilutions of the fermentation broths of Bacillus pumilus ACCC 01171, bacillus pumilus ACCC 01891 and Bacillus pumilus ACCC 01940, respectively, and the results are shown in Table 7 and B in FIG. 7.

TABLE 7 wheat budding Rate after treatment of each Strain under saline-alkaline conditions

Compared with other two strains of bacillus pumilus, the method has the advantages that the germination test of the plate shows that the treatment of the bacillus pumilus KN-601 can obviously improve the emergence rate of wheat seeds in a saline-alkali environment, and the emergence time is 1 day earlier than that of saline-alkali stress; as shown by the potted rice test, compared with other three strains of bacillus pumilus, the treatment of the bacillus pumilus KN-601 can obviously improve the emergence of rice under the saline-alkali stress.

Example 5: colonisation ability of Bacillus pumilus KN-601 in saline-alkali soil

Sterilizing saline-alkali soil sample (pH 8.9, total salt content 0.24%) collected from Ningxia at 121deg.C for 40min, and irrigating Bacillus pumilus KN-601 fermentation broth (diluted to spore number of 1×10) with 30% of the mass of the sterilized soil sample ⁹ CFU/mL), the initial value of bacillus pumilus KN-601 spores in soil is measured on the same day of irrigation, soil samples 7 cm away from the soil surface are taken 1 time every 3 days, the change of the spores in the soil is measured, sampling is continuously carried out for 6 times, and watering is carried out periodically according to 30% of the soil mass during the measurement period, so that the soil is kept moist. In the test, bacillus pumilus ACCC 01171 is set as a comparison groupThe test method is the same as above. The measurement results are shown in Table 8.

TABLE 8 colonization of Bacillus pumilus KN-601 in saline-alkali soil

As shown by the test results, the Bacillus pumilus KN-601 can survive and obviously proliferate after being applied to saline-alkali soil, and the spore number of the Bacillus pumilus in the soil is increased to 1.89 multiplied by 10 after 6 days of application ⁸ CFU/g, the spore count in soil can still reach 2.32X10 after 15 days of application ⁶ CFU/g; in contrast, the Bacillus pumilus of the control group showed a decrease in spore count without increasing after application to the soil, and the spore count in the soil was only 4.58×10 at 15 days after application ⁴ CFU/g, significantly lower than B.pumilus KN-601 treated group.

Example 6: bacillus pumilus KN-601 liquid preparation and preparation method thereof

The concentration of the strain is 2×10 by using industrial sulfuric acid or hydrochloric acid ¹⁰ Acidifying the fermentation broth of the CFU/mL bacillus pumilus KN-601, and adjusting the pH of the fermentation broth to 4.2 (pH electrode); then filtering by using a 1000-mesh vibrating screen of the high-speed mechanical Co., ltd, wherein the filtering screen is a 100-mesh screen, and filtering to obtain KN-601 filtrate; adding 0.4% sodium benzoate (w/w) into the filtrate, mechanically stirring for 30min, and adding industrial sulfuric acid or hydrochloric acid to re-regulate pH to 4.2; adding 0.15% xanthan gum (w/w%), mechanically stirring for 60min, and stopping stirring to obtain strain with concentration of 1×10 ¹⁰ CFU/mL liquid formulation of Bacillus pumilus KN-601.

Example 7: compound microbial preparation containing bacillus pumilus KN-601 and preparation thereof

The microbial preparation provided by the embodiment contains bacillus amyloliquefaciens KN-530, bacillus pumilus KN-601 and bacillus licheniformis KN-403, and the spore number ratio of the bacillus amyloliquefaciens KN-530 to the bacillus pumilus KN-403 is 5:3:2. The present example uses a strain concentration of 1X 10 ¹² CFU/g bacillus amyloliquefaciens KN-530 raw powder with strain concentration of 1 multiplied by 10 ¹² CFU/g Bacillus pumilus KN-601 raw powder with strain concentration of 1×10 ¹² The preparation method of the microbial preparation from CFU/g bacillus licheniformis KN-403 raw powder comprises the following steps:

mineral source humic acid and humus are mixed according to the mass ratio of 1:1, adding calcium phosphate with the mass fraction of 1% and bacillus subtilis fermentation residues with the mass fraction of 5% by taking the total mass of mineral humic acid and humus as a raw material, uniformly mixing, adding a granulating adhesive with the mass fraction of 2% (the adhesive is conventional in industry and can be purchased in market) and water with the mass fraction of 15%, preparing particles by adopting a disc granulating mode, and drying the particles by a drying bed to obtain an organic particle finished product (the organic content of the organic particles is more than 45% and the water content is 18%); adding the prepared organic matter particles into a fertilizer coating barrel, adding mixed raw powder (mixed raw powder according to a spore ratio, wherein the mixed raw powder has a mass fraction of 0.18 percent (based on the mass of the organic matter particles), the mixing raw powder has a problem of detection rate when coated on the particles due to a certain bacterial powder loss in the coating process, the feeding amount is slightly higher than a theoretical value), adding an adhesive protective agent (the adhesive protective agent is a solution of sodium alginate with a mass fraction of 0.1 percent) with a mass fraction of 0.5 percent (based on the mass of the organic matter particles), drying the bacterial agent particles by using a blower (the drying temperature is 60 ℃), uniformly wrapping the bacterial powder, and drying the bacterial powder until the moisture reaches 16 percent to obtain the effective viable bacterial number of more than or equal to 1 multiplied by 10 ⁹ CFU/g complex microbial formulation.

Example 8: compound microbial preparation containing bacillus pumilus KN-601 and preparation method thereof

The microbial preparation provided in this example contains Bacillus pumilus KN-601 and Paenibacillus polymyxa KN-03, and the spore ratio of the two is 8:2. The present example uses a strain concentration of 1X 10 ¹² CFU/g Bacillus pumilus KN-601 raw powder with strain concentration of 1×10 ¹¹ The preparation method of the microbial preparation from the paenibacillus polymyxa KN-03 raw powder with the CFU/g comprises the following steps:

uniformly mixing mineral source humic acid, KN-601 fermentation liquor fungus dreg and kaolin (the mass ratio of auxiliary materials is 1:1:8), and then adding 0.6% of bacillus pumilus into the uniformly mixed auxiliary materialsAdding 1% adhesive (one or more of sodium alginate, sodium hydroxymethyl cellulose and chitosan) into 1.5% KN-601 raw powder and 1.5% Paenibacillus polymyxa KN-03 raw powder, adding 10% tap water, mixing, granulating with extrusion granulator, vibration drying at 60-80deg.C to dry the water to 8% to obtain effective viable bacteria number of 1×10 ¹⁰ CFU/g complex microbial formulation.

Example 9: compound microbial preparation containing bacillus pumilus KN-601 and preparation method thereof

The microbial preparation provided by the embodiment contains bacillus pumilus KN-601 and trichoderma citrinoviride KN-T108, and the spore ratio of the bacillus pumilus KN-601 to the trichoderma citrinoviride KN-T108 is 9:1.

The trichoderma citrinoviride spore powder is obtained according to the following method:

(1) Activating strains: inoculating Trichoderma citrinoviride KN-T108 strain stored in an ultralow temperature refrigerator on a PDA flat plate, and culturing in a 28 ℃ constant temperature incubator for 3-5 days in an inverted manner;

(2) Preparing a secondary liquid strain: preparing a PDB culture medium, carrying out wet heat sterilization at 121 ℃ for 30 minutes, inoculating activated KN-T108 strain into the sterilized PDB culture medium, placing the culture medium into a constant-temperature shaking table, controlling the temperature to 28 ℃, and carrying out liquid fermentation at the rotation speed of 160 revolutions per minute for 3-4 days to obtain a large number of flocculent hypha and conidium bacterial suspension secondary strain;

(3) Solid fermentation of trichoderma: 600-800g of moso bamboo powder, 100-200g of bran, 100-200g of bean pulp, 50-100g of maltodextrin, 30-50g of soybean oil and 900-1500mL of sterile water are taken, stirred and mixed uniformly, put into a sterilizing pot for sterilization at 121 ℃ for 30 minutes, taken out, added with 1000mL of secondary liquid strain, fully stirred by using a sterilized stirring rod, and put into a 28 ℃ incubator for constant-temperature culture for 12-15 days until all the materials become green;

(4) Collecting spore powder: spreading the solid fermentation product, air-drying at room temperature for 2-3 days to evaporate water, and collecting spore powder with Trichoderma citrinoviride spore number of 3×10 by cyclone ¹⁰ Spores/g.

The present example uses a strain concentration of 1X 10 ¹² CFU/g Bacillus pumilus KN-601 raw powder with spore concentration of 3×10 ¹⁰ The preparation method of the microbial preparation from the spore/g Trichoderma citrinoviride spore powder is the same as that of example 8, except that 0.6% (w/w%) of Bacillus pumilus KN-601 raw powder and 2% (w/w%) of Trichoderma citrinovacie spore powder are added into auxiliary materials.

Example 10: application of compound microbial preparation in improving rice yield in saline-alkali soil

The effective viable count obtained in example 7 is not less than 1×10 ⁹ The CFU/g composite microbial preparation is used for rice field experiments, the experiments are carried out in saline-alkali soil (pH 7.9, soil total salt content 0.28%) in Ningxia Pingro county in 2022, 2 treatments are set for the experiments, and the treatment areas of the composite microbial preparation and the conventional treatment areas of farmers, specifically, the treatment areas are respectively applied with 20 kg/mu of the composite microbial preparation and 20 kg/mu of the compound fertilizer (17-17-17), and the conventional treatment areas are applied with 25 kg/mu of the compound fertilizer (17-17). Soil preparation after fertilization, sowing rice seeds, investigation of the emergence rate 15-20 days after emergence of rice, and the results are shown in Table 9 and FIG. 8.

Table 9 product usage in saline-alkaline paddy field

When topdressing is applied in the rice jointing period, 5 kg/mu of compound microbial agent is applied to the treatment area, 5 kg/mu of urea is applied to the conventional treatment area, the yield is measured during harvesting, 1243.6 jin of mu of the treatment area is achieved, 924.9 jin of mu of the conventional treatment area is achieved, and the yield of the treatment area is increased by 34.45% compared with that of the conventional treatment area.

Example 11: application of compound microbial preparation in improving wheat yield in saline-alkali soil

The effective viable count obtained in example 8 is not less than 1×10 ¹⁰ The CFU/g composite microbial preparation is used for wheat field test, and the test is carried out in 10 months in 2022 on severe saline-alkali soil (pH 8.6 and soil total salt content 0) in eastern mountain areas.62%) and 2 treatments were set up for the test, which were the composite microbial agent treatment area (2 kg/mu) and the farmer's conventional management area, respectively. The composite microbial preparation in the treatment area was co-sown with seeds, and the quality of wheat seedlings before winter was investigated 60 days after wheat emergence, and the results are shown in Table 10.

When the wheat is turned green in 2023, the bacillus pumilus KN-601 liquid preparation obtained in the example 6 is continuously sprayed, the mu consumption is 100mL, 500mL of liquid nitrogen fertilizer is sprayed in a control area, the yield is measured when the wheat is harvested, and the specific results are shown in Table 10.

Table 10 wheat seedling quality before winter and yield at harvest questionnaire

As is clear from the above test results, the effective viable count is not less than 1X 10 ¹⁰ The CFU/g composite microbial preparation can improve the quality of seedlings before wheat in saline-alkali soil and winter, and the wheat in the eastern camping land is frozen due to the low temperature in spring of 2023, but the wheat in the test treatment area grows normally (see figure 9), which shows that the microbial agent containing bacillus pumilus KN-601 can improve the tolerance of the wheat to low temperature and prevent and treat the occurrence of freezing injury.

Example 12: application of liquid preparation obtained in example 6 in improving potato yield in saline-alkali soil

The strain prepared in example 6 was found to have a concentration of 1X 10 ¹⁰ The CFU/mL bacillus pumilus KN-601 liquid preparation is used for potato field experiments, the experiments are carried out in saline-alkali soil (pH 8.2 and soil total salt content 0.44%) such as Shanxi elm in 2022, and 2 treatments are set in the experiments, namely a KN-601 liquid preparation treatment area and a peasant household conventional management area. The treatment area is applied with 1000 mL/mu KN-601 liquid preparation in the potato tuber forming period (bud to initial flowering period), then 1000 mL/mu KN-601 liquid preparation in the potato tuber expanding period, and the yield is measured during harvesting. The per mu yield of the potatoes in the treatment area is 6.67 tons, the commodity potato rate is 81.5 percent, the yield is increased by 26.6 percent compared with that of the potatoes in the conventional management area, the treatment area can prevent premature senility, the potato plants in the treatment area are still greenish when being harvested, and the plants in the conventional management area start to dry upDeath.

Example 13: example 8 application of microbial preparation to promotion of cowpea production in non-saline-alkaline land

The effective viable count obtained in example 8 is not less than 1×10 ¹⁰ The CFU/g compound microorganism preparation is used for cowpea field test, the test is carried out in Shandong county on 1 month 2 days of 2023, and 2 treatments are set for the test, wherein the dosage of the compound microorganism preparation treatment area is 2 kg/mu and the conventional management area of farmers. The compound microorganism preparation is applied in holes when cowpea is transplanted, the conventional management area is applied with the commercial microorganism microbial inoculum (10 hundred million/g bacillus subtilis) with the dosage of 10 kg/mu, and the growth condition of cowpea is investigated 55 days after cowpea transplanting, and the specific table is shown in Table 11 and FIG. 10.

Table 11 cowpea plant growth trait questionnaire

As shown by the test results, the plants in the treatment area are 20cm higher than those in the conventional management area, and the cowpea leaves are greenish, large in leaf, short in internode and free from overgrowth, which indicates that the effective viable count is more than or equal to 1 multiplied by 10 ¹⁰ The CFU/g composite microbial preparation still has better functions of promoting growth and early flowering when being used in a non-saline-alkaline environment.

Example 14: application of compound microbial preparation in promoting wheat production in non-saline-alkali soil

The compound microorganism preparation prepared in example 9 is used for wheat field test, the test is carried out in Shandong coast state in 2022 month 11, and 3 treatments are set for the test, namely a compound microorganism preparation treatment area (the dosage is 2 kg/mu), a trichoderma citrinoviride KN-T108 granule (2 kg/mu) treatment area with 10 hundred megaspores/gram and a peasant household conventional management area. The application method is that the microbial preparation and seeds are sown simultaneously, wheat seedling quality before winter is investigated 60 days after wheat emergence, and the disease condition of wheat stem basal rot is investigated at the bottom of 3 months of the next year, and the results are shown in Table 12.

The investigation method of the disease condition of the wheat stem rot refers to the 16 th part of pesticide field efficacy test criterion: the bactericide can prevent and cure wheat root rot.

The preparation method of the 10 hundred million spores/gram trichoderma citrinoviride KN-T108 granule comprises the following steps: will be 3X 10 ¹⁰ 40g of spore/g trichoderma citrinoviride mother powder (because of a certain detection rate, the actual feeding amount is slightly higher than a theoretical value), 530g of kaolin, 180g of corn starch, 100g of mica powder, 50g of humic acid and 10g of polyglutamic acid are uniformly mixed, 150g of sodium alginate solution (10 g is dissolved in 140g of tap water), after uniform mixing, the mixture is granulated by an extrusion granulator, after granulating and finishing, the granules are subjected to a vibration drying bed, the temperature of the drying bed is 45 ℃, and the moisture of the granules is dried to 8%, thus finishing the preparation of the granules.

Table 12 wheat seedling quality and Stem rot questionnaire before winter

The test result shows that the seedling quality before the wheat in the treatment area is obviously superior to that in the conventional management area, the tiller number is more than 3, and the tiller number in the conventional management area is only 1.9. Although the trichoderma citrinoviride KN-T108 has the function of preventing and treating soil-borne diseases, the compound use of the bacillus pumilus KN-601 and the trichoderma citrinoviride KN-T108 can improve the prevention and treatment effect of the trichoderma citrinoviride KN-T108 on the wheat stem rot, the prevention and treatment effect reaches 75.28 percent, and the prevention and treatment effect of single 10 hundred megaspores/gram trichoderma citrinoviride KN-T10 particles on the wheat stem rot is only 50.79 percent.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Bacillus pumilus KN-601 is characterized in that the preservation number is CCTCC NO: m20221316.

2. Use of bacillus pumilus KN-601 according to claim 1 for increasing the resistance of crops to abiotic stress.

3. The use according to claim 2, wherein the abiotic stress is a low temperature stress, a salt-alkali stress, an acid-alkali stress or an oligotrophic stress.

4. The use of bacillus pumilus KN-601 according to claim 1 for increasing seed emergence rate under salt and alkali stress.

5. The use of bacillus pumilus KN-601 according to claim 1 for promoting crop growth and increasing crop yield under non-stress conditions.

6. A microbial agent comprising Bacillus pumilus KN-601 according to claim 1.

7. The microbial agent according to claim 6, wherein the microbial agent is a single-dose product of bacillus pumilus KN-601 or a compound product prepared from bacillus pumilus KN-601 and any one or more of bacillus, paenibacillus and fungus.

8. A biological bacterial fertilizer comprising bacillus pumilus KN-601 of claim 1.

9. Use of the microbial agent of any one of claims 6 or 7 or the biological bacterial fertilizer of claim 8 for improving the abiotic stress resistance of crops, promoting plant growth and improving crop yield.

10. The use according to claim 9, wherein the abiotic stress is a low temperature stress, a salt-alkali stress, an acid-alkali stress or an oligotrophic stress.