Bacillus atrophaeus LYZ1127, biocontrol agent and its application

Abstract

The present application provides Bacillus atrophaeus LYZ1127, a biocontrol agent and its application. For the Bacillus atrophaeus LYZ1127, the preservation number of the Bacillus atrophaeus LYZ1127 is CGMCC No. 31718. This strain of the present application has good antibacterial activities against 11 pathogenic bacteria of 6 common diseases of alfalfa and Verticillium dahliae (isolated from Oxytropis ochrocephala plants) which is of the same genus but different species from Verticillium albo-atrum. Through pot experiments, it can be known that Bacillus atrophaeus LYZ1127 can promote the growth of alfalfa roots and plants, and can also significantly reduce the incidence of alfalfa verticillium wilt, showing a good control effect on alfalfa verticillium wilt, providing an important theoretical basis for the biological control of alfalfa verticillium wilt.

Description

Technical Field

[0001] The present application relates to the technical field of plant disease control, and particularly to Bacillus atrophaeus LYZ1127, a biocontrol agent, and its application. Background Art

[0002] Verticillium alfalfae is a quarantine and destructive soil-borne disease, which seriously damages the productivity of alfalfa. This disease not only causes a sharp decline in alfalfa yield, but also shortens the service life of alfalfa grasslands, resulting in serious economic losses and having a negative impact on the ecology and environment.

[0003] Biocontrol bacteria are widely used to control plant diseases, such as root rot, fusarium wilt, etc. They effectively inhibit the growth and spread of pathogenic bacteria and protect the healthy growth of plants through mechanisms such as antagonism, competition, and induced resistance. However, no biocontrol bacteria for controlling Verticillium alfalfae have been found yet. Summary of the Invention

[0004] In view of this, the purpose of the present application is to provide Bacillus atrophaeus LYZ1127, a biocontrol agent, and its application, so as to solve the problem that no biocontrol strain with biological control effect on Verticillium alfalfae has been found in the prior art.

[0005] The technical solution adopted by the present application to solve the above technical problems is as follows:

[0006] An embodiment of the present invention provides Bacillus atrophaeus LYZ1127, and the preservation number of the Bacillus atrophaeus LYZ1127 is CGMCC No. 31718.

[0007] In some embodiments, the 16S rDNA sequence of the Bacillus atrophaeus LYZ1127 is as shown in SEQ ID No: 1 in the sequence listing.

[0008] In some embodiments, the Bacillus atrophaeus LYZ1127 has the function of protein degradation; and / or

[0009] the Bacillus atrophaeus LYZ1127 has the function of nitrogen fixation; and / or

[0010] the Bacillus atrophaeus LYZ1127 has the function of potassium release.

[0011] Correspondingly, an embodiment of the present invention provides a biocontrol agent, and the biocontrol agent includes the Bacillus atrophaeus LYZ1127.

[0012] In some embodiments, the biocontrol agent further includes excipients; and / or

[0013] The viable count of Bacillus atrophaeus LYZ1127 in the biocontrol agent is 10 5 CFU / mL to 10 10 CFU / mL.

[0014] Correspondingly, an embodiment of the present invention provides the application of the described biocontrol agent in preventing and controlling alfalfa diseases, and the alfalfa diseases are one or more of alfalfa verticillium wilt, alfalfa root rot, alfalfa phoma leaf spot, alfalfa anthracnose, alfalfa stemphylium leaf spot, and alfalfa Cangzhou leaf blight.

[0015] In some embodiments, the pathogen of alfalfa verticillium wilt is Verticillium alfalfae LYZ0257;

[0016] The pathogens of alfalfa root rot are one or more of Paraphoma radicina LYZ0187, Fusarium oxysporum LYZ0357, Fusarium solani LYZ0358, Paraphoma chrysanthemicola LYZ0368, Plectosphaerella cucumerina LYZ0547, and Fusarium tricinctum LYZ0551; the pathogen of alfalfa phoma leaf spot is Phoma medicaginis LYZ0429; the pathogen of alfalfa anthracnose is Colletotrichum americae-borealis LYZ0544; the pathogen of alfalfa stemphylium leaf spot is Stemphylium botryosum LYZ0550; the pathogen of alfalfa Cangzhou leaf blight is Slafractonia leguminicola LYZ0582.

[0017] In some embodiments, the Oxytropis ochrocephala disease is Oxytropis ochrocephala verticillium wilt; the pathogen of Oxytropis ochrocephala verticillium wilt is Verticillium dahliae LYZ0382.

[0018] Correspondingly, an embodiment of the present invention provides the application of the described biocontrol agent in promoting the growth of alfalfa.

[0019] Correspondingly, an embodiment of the present invention provides a disease-resistant agent, and the disease-resistant agent includes the Bacillus atrophaeus LYZ1127.

[0020] In some embodiments, the disease-resistant agent is a disease-resistant agent for alfalfa diseases caused by Verticillium alfalfae LYZ0257, Paraphoma radicina LYZ0187, Fusarium oxysporum LYZ0357, Fusarium solani LYZ0358, Paraphoma chrysanthemicola LYZ0368, Phoma medicaginis LYZ0429, Colletotrichum americae-borealis LYZ0544, Plectosphaerella cucumerina LYZ0547, Stemphylium botryosum LYZ0550, Fusarium tricinctum LYZ0551, or Slafractonia leguminicola LYZ0582; and / or

[0021] The disease-resistant agent is a disease-resistant agent for Oxytropis ochrocephala diseases caused by Verticillium dahliae LYZ0382.

[0022] In this application, a bacterium, Bacillus atrophaeus LYZ1127, was isolated from alfalfa stems. It was identified as Bacillus atrophaeus LYZ1127 by morphological and molecular biological methods and was deposited with the deposit number CGMCC No. 31718. This strain has good antibacterial activity against 11 pathogenic bacteria of 6 common alfalfa diseases and Verticillium dahliae (isolated from Oxytropis ochrocephala plants) which is of the same genus but different species as Verticillium alfalfae. Through pot experiments, it was found that Bacillus atrophaeus LYZ1127 can promote the growth of alfalfa roots and plants, and can also significantly reduce the incidence of alfalfa verticillium wilt, showing good control effect on alfalfa verticillium wilt, providing an important theoretical basis for the biological control of alfalfa verticillium wilt. Brief Description of the Drawings

[0023] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description only relate to some embodiments of this application and do not limit this application, where:

[0024] Figure 1Colony characteristics and Gram-stained spore characteristics of Bacillus atrophaeus LYZ1127 strain of the present application grown on the surface of LB medium for 72 hours;

[0025] Figure 2 Phylogenetic tree of Bacillus atrophaeus LYZ1127 strain and related strains of the present application;

[0026] Figure 3 Antibacterial colony characteristics of Bacillus atrophaeus LYZ1127 strain of the present application against 11 common pathogenic bacteria of alfalfa diseases and 1 pathogenic bacterium of Oxytropis ochrocephala disease;

[0027] Figure 4 Effect diagrams of protein hydrolysis, nitrogen fixation, and potassium solubilization of Bacillus atrophaeus LYZ1127 of the present application;

[0028] Figure 5 Effect diagrams of root growth promotion of alfalfa plants and prevention and control of alfalfa verticillium wilt by Bacillus atrophaeus LYZ1127 of the present application;

[0029] Figure 6 Effect diagrams of growth promotion of alfalfa plants and prevention and control of alfalfa verticillium wilt by Bacillus atrophaeus LYZ1127 of the present application;

[0030] Figure 7 Effect of Bacillus atrophaeus LYZ1127 of the present application on the plant height of alfalfa;

[0031] Figure 8 Effect of Bacillus atrophaeus LYZ1127 of the present application on the number of compound leaves of alfalfa;

[0032] Figure 9 Effect of Bacillus atrophaeus LYZ1127 of the present application on the number of branches of alfalfa;

[0033] Figure 10 Effect of Bacillus atrophaeus LYZ1127 of the present application on the diameter of the main stem of alfalfa;

[0034] Figure 11 Effect of Bacillus atrophaeus LYZ1127 of the present application on the root length of alfalfa;

[0035] Figure 12 Effect of Bacillus atrophaeus LYZ1127 of the present application on the diameter of the main root of alfalfa;

[0036] Figure 13 Effect of Bacillus atrophaeus LYZ1127 of the present application on the fresh weight per plant of alfalfa;

[0037] Figure 14 Effect of Bacillus atrophaeus LYZ1127 of the present application on the dry weight per plant of alfalfa;

[0038] Figure 15 Effect of Bacillus atrophaeus LYZ1127 of the present application on the incidence rate of alfalfa

[0039] Figure 16 Effect of Bacillus atrophaeus LYZ1127 of the present application on the disease index of alfalfa

[0040] Figure 17 Control effect diagram of Bacillus atrophaeus LYZ1127 of the present application on alfalfa Detailed implementation manners

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present application. In addition, it should be understood that the specific implementation manners described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0042] In the present application, unless otherwise stated, the orientation terms such as "upper" and "lower" generally refer to the upper and lower in the actual use or working state of the device, specifically the drawing direction in the drawings; and "inner" and "outer" refer to the outline of the device. In addition, in the description of the present application, the term "including" means "including but not limited to". The terms first, second, third, etc. are only used as labels and do not impose numerical requirements or establish an order.

[0043] In the present application, "and / or" describes the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural.

[0044] In the present application, "at least one" means one or more, and "multiple" means two or more. "One or several", "at least one (item) below" or similar expressions refer to any combination of these items, including any combination of single item (item) or plural items (items). For example, "at least one (item) of a, b, or c", or, "at least one (item) of a, b, and c" can all represent: a, b, c, a-b (that is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple respectively.

[0045] The various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a rigid limitation on the scope of the present application; therefore, it should be considered that the described range description has specifically disclosed all possible sub-ranges and individual values within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0046] In a first aspect, an embodiment of the present invention provides Bacillus atrophaeus LYZ1127, and the preservation number of the Bacillus atrophaeus LYZ1127 is CGMCC No. 31718.

[0047] In this application, a strain of bacteria, Bacillus atrophaeus LYZ1127, was isolated from alfalfa stems. It was identified as Bacillus atrophaeus LYZ1127 through morphological and molecular biological methods and was preserved with the preservation number CGMCC No. 31718. This strain has good antibacterial activity against 11 pathogenic bacteria of 6 common diseases of alfalfa and Verticillium dahliae (isolated from Oxytropis ochrocephala plants) which is of the same genus but different species from Verticillium alfalfae. Through pot experiments, it can be known that Bacillus atrophaeus LYZ1127 can promote the growth of alfalfa roots and plants, and can also significantly reduce the incidence of alfalfa verticillium wilt, showing a good control effect on alfalfa verticillium wilt, providing an important theoretical basis for the biological control of alfalfa verticillium wilt.

[0048] In this application, Bacillus atrophaeus LYZ1127 is preserved in the China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, with the preservation number CGMCC No. 31718, the preservation date is August 22, 2024, the biological material (strain): LYZ1127, and the taxonomic name: Bacillus atrophaeus.

[0049] In some embodiments, the 16S rDNA sequence of the Bacillus atrophaeus LYZ1127 is as shown in

[0050] Table SEQ ID No: 1.

[0051] In some embodiments, the Bacillus atrophaeus LYZ1127 has a protein-degrading effect.

[0052] In some embodiments, the Bacillus atrophaeus LYZ1127 has a nitrogen-fixing effect.

[0053] In some embodiments, the Bacillus atrophaeus LYZ1127 has a potassium-releasing effect.

[0054] In some embodiments, the Bacillus atrophaeus LYZ1127 is derived from the rhizosphere soil of alfalfa (Gannong No. 4).

[0055] It can be understood that the rhizosphere soil of alfalfa (Gannong No. 4); the collection location is: the breeding test field for resistant varieties of alfalfa verticillium wilt in Liuxincun, Minle County, Zhangye City, Gansu Province.

[0056] It can be understood that the alfalfa variety Gannong No. 4 has good resistance to alfalfa verticillium wilt ((V. alfalfae) LYZ0257).

[0057] In a second aspect, embodiments of the present invention provide a biocontrol agent, which includes the Bacillus atrophaeus LYZ1127.

[0058] In some embodiments, the biocontrol agent further includes excipients, and the excipients include at least one of sterile water and LB

[0059] culture medium.

[0060] Further, the viable count of Bacillus atrophaeus LYZ1127 in the biocontrol agent is 10 5 CFU / mL to 10 10 CFU / mL, for example, it can be 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, 10 10 CFU / mL, etc. Within the range of the viable count, there is a good biocontrol effect.

[0061] In a third aspect, embodiments of the present invention provide the application of the biocontrol agent in preventing and controlling alfalfa diseases, and the alfalfa diseases are one or more of alfalfa verticillium wilt, alfalfa root rot, alfalfa phoma leaf spot, alfalfa anthracnose, alfalfa stemphylium leaf spot, and alfalfa Cangzhou leaf blight.

[0062] Furthermore, the pathogen of alfalfa verticillium wilt is Verticillium alfalfae LYZ0257; the pathogens of alfalfa root rot are one or more of Paraphoma radicina LYZ0187, Fusarium oxysporum LYZ0357, Fusarium solani LYZ0358, Paraphoma chrysanthemicola LYZ0368, Plectosphaerella cucumerina LYZ0547, and Fusarium tricinctum LYZ0551; the pathogen of alfalfa phoma leaf spot is Phoma medicaginis LYZ0429; the pathogen of alfalfa anthracnose is Colletotrichum americae-borealis LYZ0544; the pathogen of alfalfa stemphylium leaf spot is Stemphylium botryosum LYZ0550; the pathogen of alfalfa Cangzhou leaf blight is Slafractonia leguminicola LYZ0582.

[0063] In a fourth aspect, an embodiment of the present invention provides an application of the biocontrol agent in promoting the growth of alfalfa.

[0064] Furthermore, the biocontrol agent promotes the growth of alfalfa roots.

[0065] More specifically, the growth of alfalfa roots includes an increase in the length of the main root and an increase in the diameter of the main root.

[0066] Furthermore, the biocontrol agent promotes the growth of alfalfa plants.

[0067] More specifically, the growth of alfalfa plants includes an increase in the plant height of alfalfa plants, an increase in the number of compound leaves, an increase in the number of branches, an increase in the diameter of the main stem, an increase in the fresh weight per plant, and an increase in the dry weight per plant.

[0068] In a fifth aspect, an embodiment of the present invention provides an application of the biocontrol agent in controlling the diseases of Oxytropis ochrocephala, and the disease of Oxytropis ochrocephala is Oxytropis ochrocephala verticillium wilt.

[0069] Furthermore, the pathogen of Oxytropis ochrocephala verticillium wilt is Verticillium dahliae LYZ0382.

[0070] In a sixth aspect, an embodiment of the present invention provides a disease-resistant agent, and the disease-resistant agent includes the Bacillus atrophaeus LYZ1127.

[0071] Furthermore, the disease-resistant agent is a disease-resistant agent for alfalfa diseases caused by Verticillium alfalfae LYZ0257, Paraphoma radicina LYZ0187, Fusarium oxysporum LYZ0357, Fusarium solani LYZ0358, Paraphoma chrysanthemicola LYZ0368, Phoma medicaginis LYZ0429, Colletotrichum americae-borealis LYZ0544, Plectosphaerella cucumerina LYZ0547, Stemphylium botryosum LYZ0550, Fusarium tricinctum LYZ0551 or Slafractonia leguminicola LYZ0582.

[0072] Furthermore, the disease-resistant agent is a disease-resistant agent for Oxytropis ochrocephala diseases caused by Verticillium dahliae LYZ0382.

[0073] Test Example 1

[0074] Morphological identification:

[0075] For the identification of cultural characteristics, the Bacillus atrophaeus LYZ1127 of the present application was streaked into single colonies on an LB medium, cultured at 28 °C, and its colony characteristics were continuously observed.

[0076] Gram staining: The Bacillus atrophaeus LYZ1127 of the present application was Gram stained after being cultured at 28 °C for 18 h. A small amount of Bacillus atrophaeus LYZ1127 was picked up with an inoculation loop and evenly smeared on a glass slide with distilled water dropped on it, and fixed three times through a flame. Crystal violet was added and stained for 1 min, rinsed with water until colorless, then iodine solution was added and stained statically for 1 min, rinsed with water until colorless. 95% alcohol was added dropwise, the glass slide was shaken, and decolorization was carried out for 20 - 60 s. After rinsing with water and blotting dry, safranin was used for staining for 1 min, rinsed with water, air-dried, and then the individual morphological characteristics were observed with an oil immersion microscope.

[0077] The results of morphological observation showed that when the strain LYZ1127 was cultured at 28 °C for 72 h, the colony was grayish white on the LB medium, circular, smooth, and had a neat edge ( Figure 1 A), Gram staining was G+, and the bacterium was observed to be rod-shaped, about 2-4 μm in length ( Figure 1 B).

[0078] Among them, the LB medium: 10.0 g of tryptone, 5.0 g of yeast extract, 5.0 g of NaCl, 1 L of distilled water.

[0079] Test Example 2

[0080] Molecular biological identification:

[0081] The single colony of Bacillus atrophaeus LYZ1127 was streaked and purified on the nutrient agar medium (NA). Then, a single colony was picked and used with the Ezup column bacterial genomic DNA extraction kit (B518255-0050) produced by Shanghai Biotechnology Co., Ltd. The concentration and quality of DNA were measured with a micro-spectrophotometer. Then, the qualified DNA samples were stored in a -20 °C refrigerator for PCR amplification.

[0082] PCR amplification, the primers used were the common primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′. The PCR system was: 5 μL of DNA template, 25 μL of 2×Tap PCR Master Mix, 1 μL of each forward and reverse primer, 18 μL of ddH2O. The PCR amplification conditions were: pre-denaturation at 95 °C for 4 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 35 cycles; extension at 72 °C for 10 min. The PCR amplification products were detected by 1.0% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0083] Tree construction, the sequencing results were compared with the sequences in the EzBioCloud database, and then the phylogenetic tree was constructed using the MEGA7.0 software to determine the taxonomic status of the strain. All sequences were connected into multi-gene fragments using the SequenceMan software. The support strength of each branch of the phylogenetic tree was evaluated by the bootstrap value of 1000 repeated bootstrap tests.

[0084] Among them, the nutrient agar medium (Nutrient Agar, NA): 10.0 g of peptone, 3.0 g of beef extract, 5.0 g of sodium chloride, 15.0 g of agar, 1 L of distilled water, and the pH value was adjusted to 7.0-7.2 with NaOH.

[0085] According to the constructed phylogenetic tree, we found that the antagonistic bacterium LYZ1127 clustered with Bacillus atrophaeus (accession number DQ993677.1) with a bootstrap value of 99%, and had relatively small bootstrap values with other species. Therefore, it could be identified as Bacillus atrophaeus. Figure 2),The sequencing sequence, i.e., SEQ ID No:1, is GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACACCCCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAGACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGT。

[0086] Based on the results of comprehensive morphological and molecular biological identification, the strain LYZ1127 was identified as Bacillus atrophaeus.

[0087] The sequence of SEQ ID No:1 above was submitted as an electronic sequence listing file that complies with the WIPOST.26 standard.

[0088] Test Example 3

[0089] Detection of antibacterial activity:

[0090] The antibacterial activity of Bacillus atrophaeus LYZ1127 was determined by the plate confrontation method. Each test pathogen was activated and cultured on a PDA plate for 3 - 5 days, and then a bacterial disc (5 mm in diameter) was punched and inoculated in the center of the PDA medium plate. The bacterial solution of Bacillus atrophaeus LYZ1127 activated in the LB liquid medium was smeared at a distance of 4 cm to the left and right of the center point of the plate, and then cultured in the dark at 28 °C for 7 days (the growth rates of different pathogens are different, and the measurement was carried out when the control group covered the culture dish). Inoculating the pathogen on the PDA medium plate was used as the blank control. Each treatment had 3 replicates, and the experiment was independently repeated 3 times. When the mycelia of each pathogen in the blank control group covered the plate, the diameter of the pathogenic fungus was measured, and the relative inhibition rate was calculated according to the following formula.

[0091] Relative inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial disc diameter) × 100.

[0092] Among them, Potato Dextrose Agar (PDA): 200.0 g of potatoes, 20.0 g of glucose, 20.0 g of agar, 1 L of distilled water;

[0093] The test pathogens were fungal strains, stored in the microbial strain library of the Grassland Microbial Center of Lanzhou University, Verticillium alfalfae LYZ0257, Paraphoma radicina LYZ0187, Verticillium dahliae LYZ0382, Fusarium oxysporum LYZ0357, Fusarium solani LYZ0358, Paraphoma chrysanthemicola LYZ0368, Phoma medicaginis LYZ0429, Colletotrichum americae - borealis LYZ0544, Plectosphaerella cucumerina LYZ0547, Stemphylium botryosum LYZ0550, Fusarium tricinctum LYZ0551, Slafractonia leguminicola LYZ0582.

[0094] Figure 3Among them, A, control colony of Verticillium alfalfae; B, inhibitory colony of LYZ1127 against Verticillium alfalfae 0257; C, control colony of Phoma radicina LYZ0187; D, inhibitory colony of LYZ1127 against Phoma radicina LYZ0187; E, control colony of Fusarium oxysporum LYZ0357; F, inhibitory colony of LYZ1127 against Fusarium oxysporum LYZ0357; G, Fusarium solani LYZ0358; H, inhibitory colony of LYZ1127 against Fusarium solani LYZ0358; I, control colony of Phoma chrysanthemicola LYZ0368; J, inhibitory colony of LYZ1127 against Phoma chrysanthemicola LYZ0368; K, control colony of Verticillium dahliae LYZ0382; L, inhibitory colony of LYZ1127 against Verticillium dahliae LYZ0382; M, colony of Phoma medicaginis LYZ0429; N, inhibitory colony of LYZ1127 against Phoma medicaginis LYZ0429; O, control colony of Colletotrichum americae-borealis LYZ0544; P, inhibitory colony of LYZ1127 against Colletotrichum americae-borealis LYZ0544; Q, control colony of Physalospora cucumerina LYZ0547; R, inhibitory colony of LYZ1127 against Physalospora cucumerina LYZ0547; S, control colony of Stemphylium botryosum LYZ0550; T, inhibitory colony of LYZ1127 against Stemphylium botryosum LYZ0550; U, control colony of Fusarium tricinctum LYZ0551; V, inhibitory colony of LYZ1127 against Fusarium tricinctum LYZ0551; W, control colony of Stemphylium leguminicola LYZ0582; X, inhibitory colony of LYZ1127 against Stemphylium leguminicola LYZ0582.

[0095] The bacteriostatic rate results of Bacillus atrophaeus LYZ1127 against 11 pathogenic bacteria of alfalfa diseases and 1 pathogenic bacteria of Oxytropis ochrocephala wilt are shown in Table 1.

[0096] Table 1 Inhibitory Rates of LYZ1127 against Pathogens of 11 Common Alfalfa Diseases and 1 Pathogen of Verticillium Wilt of Oxytropis ochrocephala

[0097]

[0098] Note: Different letters in the same column indicate significant differences (P<0.05).

[0099] From Figure 3 It can be seen from Table 1 that Bacillus atrophaeus LYZ1127 has inhibitory effects on pathogens of 11 alfalfa diseases and 1 pathogen of Verticillium wilt of Oxytropis ochrocephala, and the inhibitory rates are significantly different (P<0.05). The inhibitory rate against Verticillium alfalfae LYZ0257 is 76.69% ( Figure 3 AB), Phoma radicina LYZ0187, the inhibitory rate is 55.08% ( Figure 3 CD), Verticillium dahliae LYZ0382, the inhibitory rate is 58.92% ( Figure 3 KL), Fusarium oxysporum LYZ0357, the inhibitory rate is 58.11% ( Figure 3 EF), Fusarium solani LYZ0358, the inhibitory rate is 59.41% ( Figure 3 GH), Phoma chrysanthemicola LYZ0368, the inhibitory rate is 41.47% ( Figure 3 IJ), Phoma medicaginis LYZ0429, the inhibitory rate is 71.10% ( Figure 3 MN), Colletotrichum americae-borealis LYZ054, the inhibitory rate is 66.45% ( Figure 3 OP), Phacidiopycnis cucumerina LYZ0547, the inhibitory rate is 61.56% ( Figure 3 QR), Stemphylium botryosum LYZ0550, the inhibitory rate is 56.68% ( Figure 3 ST), Fusarium tricinctum LYZ0551, the inhibitory rate is 56.68% ( Figure 3 UV), Stemphylium leguminicola LYZ0582, the inhibitory rate is 70.21% ( Figure 3 WX).

[0100] Experimental Example 4

[0101] Protein-degrading, Nitrogen-fixing and Potassium-releasing Capabilities of Bacillus atrophaeus LYZ1127

[0102] Detection of nitrogen fixation ability: First, activate Bacillus atrophaeus LYZ1127 on LB medium, and then inoculate it into nitrogen-free medium with 3 replicates for each treatment. Incubate at 28 °C for 7 days. If colonies appear on the nitrogen-free medium, transfer them to the nitrogen-free medium again and incubate at 28 °C for 7 days, and repeat the transfer 3 times. If colonies appear on the nitrogen-free medium after 3 transfers, it indicates that the strain has nitrogen fixation ability.

[0103] Identification of protein-degrading function: First, inoculate Bacillus atrophaeus LYZ1127 on skim milk powder plates and incubate at 37 °C for 1 - 2 days. Observe the appearance of clear zones, and simultaneously measure the diameter of the clear zone (D) and the diameter of the colony (d). Judge the protein-degrading ability of each strain according to the ratio of the diameter of the clear zone to the diameter of the colony (HC).

[0104] Identification of potassium-solubilizing function: First, inoculate Bacillus atrophaeus LYZ1127 on the plate of silicate bacteria medium by the method of three-zone streaking and incubate at 37 °C for 3 - 5 days. Observe whether the strain can produce smooth, transparent, oil-drop-shaped colonies with capsules.

[0105] Such as Figure 4 , Figure 4 In A, the colony characteristics of protein-degrading of LYZ1127; B, the colony characteristics of nitrogen fixation ability determination of LYZ1127; C, the colony characteristics of potassium-solubilizing ability determination of LYZ1127;

[0106] From Figure 4 it can be seen that when Bacillus atrophaeus LYZ1127 is inoculated on the surface of the special protein-degrading medium, clear zones are produced, proving that the bacterium has protein-degrading ability (HC = 138.42)( Figure 4 A); at the same time, when the bacterium is inoculated on the special nitrogen-fixing medium and colonies still grow after 3 transfers, it proves that the bacterium has nitrogen fixation function( Figure 4 B); when the bacterium is inoculated on the surface of the potassium-solubilizing medium, the strain can produce smooth, transparent, oil-drop-shaped colonies with capsules( Figure 4 C).

[0107] Test Example 5

[0108] Determination of control efficacy in pot experiments:

[0109] Preparation of spore suspension, biocontrol bacterial liquid: Inoculate Bacillus atrophaeus LYZ1127 strain into LB culture medium, incubate at 28 °C and 200 r / min in a shaking flask for 48 h, and then dilute the culture medium with sterile water to a spore concentration of 10 8CFU / mL, for standby. Pathogen spore suspension: Inject 10 mL of sterile water onto the surface of the PDA plate inoculated with the pathogen for 2 weeks respectively, then gently scrape the surface of the colony with a sterilized glass slide to make the spores on the colony surface fall off, and then transfer the spore liquid into a sterilized 50 mL centrifuge tube. Then, with the help of a hemocytometer and a microscope, adjust the spore concentration to 10 6 spores / mL, for standby.

[0110] Germination acceleration and seedling raising: After surface disinfection, the seeds of Medicago sativa cv. Xinmu No. 1 are placed on the moist filter paper (sterilized) paved with two layers of pathogen spore suspension or sterile water (control sterile water). There are 45 seeds in each dish. Inject 5 mL of pathogen spore suspension or sterile water (control sterile water, add spore suspension for treatment) into the dish every 24 h for germination acceleration, and promote germination at 25 °C for 72 h. Select the seeds with consistent germination and plant them into the flower pots filled with sterile soil, 5 plants in each pot. The seedlings are cultured in a greenhouse with the conditions of daytime temperature 24±2 °C, nighttime temperature 7±2 °C, humidity 60%, 12 h light, and 12 h darkness, and watered as needed.

[0111] Experimental treatments: There are mainly 6 treatments in the experiment, with 7 (pots) replicates for each treatment, which are (1) sterile water; (2) LB culture solution; (3) biocontrol strain LYZ1127; (4) sterile water + V. alfalfae LYZ0257; (5) LB culture solution + V. alfalfae LYZ0257; and (6) biocontrol strain LYZ1127 + V. alfalfae LYZ0257.

[0112] Inoculation method: For pathogen inoculation, the seed soaking method is used; for biocontrol bacteria, the root canning method is used. For the seedlings germinated and soaked with sterile water (treatments 1 to 3) and the seedlings germinated and soaked with the spore suspension of V. alfalfae LYZ0257 (treatments 4 to 6), after the seedlings are transplanted into the flower pots (inner diameter 14 cm) filled with sterilized soil (black charcoal soil) and grow for 7 days, the prepared biocontrol bacteria solution, water, and LB culture solution are canned into the roots, 25 mL per pot and 5 mL per plant.

[0113] Index determination: At 60 days after inoculation, measure the plant height, number of leaves, number of branches, diameter of the main stem, root length, diameter of the main root, fresh weight, dry weight, number of diseased plants, and severity of the plants, calculate the incidence rate of alfalfa verticillium wilt, disease index, and calculate the control effect. The grading standard for alfalfa verticillium wilt to record the disease severity is shown in Table 2:

[0114] Incidence rate = (number of diseased plants / total number of plants surveyed) × 100%

[0115] Disease index = Σ (disease level × number of plants at this level) / (total number of plants surveyed × 5) × 100%

[0116] Control effect (%) = (disease index of control - disease index of treatment) / disease index of control × 100

[0117] Table 2 Grading of severity of Verticillium albo - atrum of alfalfa

[0118]

[0119] Figure 5 In Fig. A, from left to right are the alfalfa roots after inoculation with biocontrol bacterium LYZ1127, LB culture solution and sterile water, respectively. Figure 5 It can be seen that: compared with the control, the root length of alfalfa inoculated only with biocontrol bacterium LYZ1127 increased significantly, indicating that biocontrol bacterium LYZ1127 is beneficial to the growth of alfalfa roots.

[0120] Figure 5 In Fig. B, from left to right are the alfalfa roots after inoculation with biocontrol bacterium LYZ1127 + Verticillium albo - atrum LYZ0257, LB culture solution + Verticillium albo - atrum LYZ0257 and sterile water + Verticillium albo - atrum LYZ0257, respectively. Figure 5 It can be seen that: after being infected by Verticillium albo - atrum LYZ0257, compared with the control, the roots of alfalfa inoculated with biocontrol bacterium LYZ1127 showed no obvious symptoms of Verticillium wilt of alfalfa, indicating that biocontrol bacterium LYZ1127 can inhibit Verticillium albo - atrum LYZ0257.

[0121] Figure 6 In Fig. A (from right to left) are the alfalfa plants after inoculation with sterile water, LB culture solution and biocontrol bacterium LYZ1127, respectively. Figure 6 It can be seen that: compared with the control, the plant height of alfalfa inoculated only with biocontrol bacterium LYZ1127 increased significantly, indicating that biocontrol bacterium LYZ1127 is beneficial to the growth of alfalfa plants.

[0122] Figure 6 In Fig. B (from right to left) are the alfalfa plants after inoculation with sterile water + Verticillium albo - atrum LYZ0257, LB culture solution + Verticillium albo - atrum LYZ0257 and biocontrol bacterium LYZ1127 + Verticillium albo - atrum LYZ0257, respectively. Figure 6 It can be seen that: after being infected by Verticillium albo - atrum LYZ0257, compared with the control, the number of alfalfa plants showing symptoms of Verticillium wilt of alfalfa inoculated with biocontrol bacterium LYZ1127 decreased significantly. For the alfalfa plants inoculated with sterile water and LB culture solution, the disease symptoms were obvious, the alfalfa leaves turned yellow, and there were typical "V" - shaped lesions at the leaf tips.

[0123] From Figures 7 to 14 It can be seen that:

[0124] Bacillus atrophaeus LYZ1127 can significantly increase the plant height, number of compound leaves, main stem diameter, main root length, main root diameter, fresh weight per plant and dry weight per plant of alfalfa plants (P<0.05), and has no significant effect on alfalfa plants in LB culture solution.

[0125] The plant heights of alfalfa plants treated with sterile water, LB culture solution and LYZ1127 are 36.5 cm, 36.8 cm and 39.7 cm respectively, the numbers of compound leaves are 16, 22 and 25 respectively, the numbers of branches are 2, 2 and 2 respectively, the main stem diameters are 1.05 mm, 1.06 mm and 1.15 mm, the root lengths are 12.49 cm, 12.49 cm and 14.19 cm respectively, the main root diameters are 1.91 mm, 1.95 mm and 2.29 mm respectively, the fresh weights per plant are 1.45 g, 1.46 g and 1.9 g respectively, and the dry weights per plant are 0.39 g, 0.41 g and 0.42 g respectively; compared with CK, the plant height increases by about 9% after treatment with LYZ1127; the number of compound leaves increases by 52%, the main stem diameter increases by 10%, the root length increases by 14%, the main root diameter increases by 20%, the fresh weight per plant increases by about 28%, and the dry weight per plant increases by about 9%. It can be seen that the biocontrol bacterium LYZ1127 has a significant effect on the growth indexes and yield of alfalfa plants.

[0126] After Verticillium albo-atrum LYZ0257 infection, the plant heights of alfalfa plants treated with sterile water, LB culture solution and LYZ1127 are 29.3 cm, 29.3 cm and 36.7 cm respectively ( Figure 7 ), the numbers of compound leaves are 18, 16 and 23 respectively ( Figure 8 ), the numbers of branches are about 2, 2 and 2 respectively ( Figure 9 ), the main stem diameters are 1.07 mm, 1.09 mm and 1.13 mm respectively ( Figure 10 ), the root lengths are 10.66 cm, 10.84 cm and 12.84 cm respectively ( Figure 11 ), the main root diameters are 1.31 mm, 1.34 mm and 1.9 mm respectively ( Figure 12 ), the fresh weights per plant are 0.92 g, 0.90 g and 1.45 g respectively ( Figure 13 ), and the dry weights per plant are 0.29 g, 0.29 g and 0.38 g respectively ( Figure 14 ); compared with CK, the plant height increases by about 25% after treatment with LYZ1127; the number of compound leaves increases by 27%, the main stem diameter increases by 6%, the root length increases by 21%, the main root diameter increases by 48%, the fresh weight per plant increases by about 57%, and the dry weight per plant increases by about 32%. It can be seen that after Verticillium albo-atrum LYZ0257 infects alfalfa plants, it has a significant effect on alfalfa plants, but the biocontrol bacterium LYZ1127 can weaken the influence of the pathogen on alfalfa plants.

[0127] From Figure 15 Figure 16 and Figure 17 It can be seen that after treatment with sterile water (CK), LB culture medium and Bacillus atrophaeus LYZ1127, the incidence rates of Verticillium wilt of alfalfa were 91.4%, 88.6% and 28.6% respectively, and the disease indices were 43.4, 42.9 and 8.0 respectively. The control effects of LB culture medium and Bacillus atrophaeus LYZ1127 on Verticillium wilt of alfalfa were 1.3% and 81.58% respectively. It can be seen that Bacillus atrophaeus LYZ1127 can significantly reduce the incidence rate of Verticillium wilt of alfalfa and has a good control effect on Verticillium wilt of alfalfa.

[0128] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only an example and does not constitute a limitation to this application. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this application. Such modifications, improvements and corrections are suggested in this application, so such modifications, improvements and corrections still fall within the spirit and scope of the exemplary embodiments of this application.

[0129] Meanwhile, this application uses specific terms to describe the embodiments of this application. Such as "one embodiment", "an embodiment", and / or "some embodiments" mean a certain feature, structure or characteristic related to at least one embodiment of this application. Therefore, it should be emphasized and noted that the "an embodiment" or "one embodiment" or "an alternative embodiment" mentioned twice or more at different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this application can be appropriately combined.

Claims

1. Bacillus atrophaeus LYZ1127, characterized in that, The preservation number of the Bacillus atrophaeus LYZ1127 is CGMCC No. 31718.

2. Biocontrol agent, characterized in that: The biocontrol agent includes the Bacillus atrophaeus LYZ1127 described in claim 1.

3. The biocontrol agent according to claim 2, wherein: The biocontrol agent further includes excipients; and / or The viable count of Bacillus atrophaeus LYZ1127 in the biocontrol agent is 10 5 CFU / mL to 10 10 CFU / mL.

4. Use of the biocontrol agent according to claim 2 in controlling alfalfa diseases, wherein the alfalfa diseases are one or more of alfalfa verticillium wilt, alfalfa root rot, Phoma medicaginis leaf spot, alfalfa anthracnose, Stemphylium alfalfae leaf spot, and alfalfa Cangzhou leaf blight.

5. Use of the biocontrol agent according to claim 2 in controlling Oxytropis ochrocephala diseases, wherein the Oxytropis ochrocephala disease is Oxytropis ochrocephala verticillium wilt.

6. Use of the biocontrol agent according to claim 2 in promoting the growth of alfalfa.

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