Bacillus subtilis LYZ1128, biocontrol inoculant and application of biocontrol inoculant

Abstract

The invention provides bacillus subtilis LYZ1128, a biocontrol microbial inoculum and application of the biocontrol microbial inoculum. The preservation number of the bacillus subtilis LYZ1128 is CGMCC (China General Microbiological Culture Collection Center) No.31719; the bacillus subtilis LYZ1128 disclosed by the invention has relatively good bacteriostatic activity on 11 pathogenic bacteria of six common diseases of alfalfa and verticillium dahliae (separated from oxytropis aureocauda plants) belonging to the same species and different species as the verticillium medicago, and pot experiment shows that the bacillus subtilis LYZ1128 can promote the growth of alfalfa root systems and plants, so that the growth of the alfalfa root systems and plants can be promoted; the morbidity of the verticillium wilt of the alfalfa can be obviously reduced, a good prevention and treatment effect on the verticillium wilt of the alfalfa is achieved, and an important theoretical basis is provided for biological prevention and treatment of the verticillium wilt of the alfalfa.

Description

Technical Field

[0001] The present application relates to the technical field of plant disease prevention and control, and in particular to Bacillus subtilis LYZ1128, biocontrol agents and applications thereof. Background Art

[0002] Alfalfa Verticillium wilt is a devastating disease for alfalfa. Its pathogen is Verticillium alfalfae, a vascular parasite that can cause damage to all tissues of alfalfa plants, and diseased branches are shorter than healthy branches. The disease is common in the Hexi Corridor of Gansu Province. The main prevention and control measures currently used are breeding disease-resistant varieties and chemical control.

[0003] So far, no varieties with good resistance to alfalfa Verticillium wilt have been screened out in China. Although chemical control can effectively control the spread of alfalfa Verticillium wilt to a certain extent, it will pollute the environment, destroy the ecosystem, and endanger human health.

[0004] Based on the shortcomings of the above prevention and control methods, it is very important to find a green and environmentally friendly prevention and control method that can prevent and control alfalfa wilt on a large area in the Hexi region. Summary of the invention

[0005] In view of this, the purpose of the present application is to provide Bacillus subtilis LYZ1128, a biocontrol agent and its application, so as to solve the problem that no biocontrol strain with biological control effect on alfalfa Verticillium alfalfae has been found in the prior art.

[0006] The technical solution adopted by this application to solve the above technical problems is:

[0007] The embodiment of the present invention provides Bacillus subtilis LYZ1128, wherein the Bacillus subtilis

[0008] The deposit number of LYZ1128 is CGMCC No.31719.

[0009] In some embodiments, the 16S rDNA sequence of the Bacillus subtilis LYZ1128 is shown in SEQ ID No: 1 in the sequence listing.

[0010] In some embodiments, the Bacillus subtilis LYZ1128 has a proteolytic effect; and / or

[0011] The Bacillus subtilis LYZ1128 has a nitrogen-fixing function; and / or

[0012] The Bacillus subtilis LYZ1128 has a potassium-dissolving effect

[0013] Accordingly, an embodiment of the present invention provides a biocontrol agent, which includes the Bacillus subtilis LYZ1128.

[0014] In some embodiments, the biocontrol agent further comprises an auxiliary material; and / or

[0015] The number of viable bacteria of Bacillus subtilis LYZ1128 in the biocontrol agent is 10 5 CFU / mL~10 10 CFU / mL.

[0016] Accordingly, an embodiment of the present invention provides an application of the biocontrol agent in preventing and controlling alfalfa diseases, wherein the alfalfa diseases are one or more of alfalfa verticillium wilt, alfalfa root rot, alfalfa stem spot mildew, alfalfa anthracnose, alfalfa stolonifer leaf spot and alfalfa Cangzhou leaf blight.

[0017] In some embodiments, the pathogen of alfalfa Verticillium wilt is Verticillium alfale LYZ0257;

[0018] 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 stem spot is Phoma medicaginis LYZ0429; the pathogen of alfalfa anthracnose is Colletotrichum americae-borealis LYZ0544; the pathogen of alfalfa stalk spot is Stemphylium stalk botryosum)LYZ0550; the pathogen of alfalfa leaf blight is Slafractonia leguminicola LYZ0582.

[0019] In some embodiments, the disease of Oxytropis erythrorhizon is Verticillium wilt of Oxytropis erythrorhizon; the pathogen of Verticillium wilt of Oxytropis erythrorhizon is Verticillium dahliae LYZ0382.

[0020] Accordingly, an embodiment of the present invention provides the use of the biocontrol agent in promoting the growth of alfalfa.

[0021] Accordingly, an embodiment of the present invention provides an anti-disease agent, which includes the Bacillus subtilis LYZ1128.

[0022] In some embodiments, the anti-disease agent is caused by V.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 solani LYZ0358, Paraphoma chrysanthemicola LYZ0368, Phoma medicaginis LYZ0429, Colletotrichum americae-borealis LYZ0544, Plectosphaerella cucumerina LYZ0547, Stemphylium botryosum LYZ0550, Fusarium tricinctum) LYZ0551 or alfalfa disease caused by Cangzhou leaf blight (Slafractonia leguminicola) LYZ0582; and / or

[0023] The disease-resistant agent is a disease-resistant agent for the disease of Oxytropis flavonoides caused by Verticillium dahliae LYZ0382.

[0024] The present application separates a bacterium, Bacillus subtilis LYZ1128, from alfalfa stems. The bacterium is identified as Bacillus subtilis LYZ1128 by morphology combined with molecular biology, and is preserved with a preservation number of CGMCC No.31719. The strain has good antibacterial activity against 11 pathogens of 6 common alfalfa diseases and Verticillium dahliae (isolated from Oxytropis flavescentis plants) of the same genus but different species as Verticillium alfalfa. Potted plant experiments show that Bacillus subtilis LYZ1128 can promote the growth of alfalfa roots and plants, and can also significantly reduce the incidence of alfalfa Verticillium wilt, and has a 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

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings of the embodiments are briefly introduced below. Obviously, the drawings described below only relate to some embodiments of the present application, but are not intended to limit the present application, wherein:

[0026] Figure 1 The colony characteristics and Gram-stained spore characteristics of the Bacillus subtilis LYZ1128 strain of this application grown on the surface of LB medium for 72 hours;

[0027] Figure 2 Phylogenetic tree of Bacillus subtilis LYZ1128 strain and related strains of this application;

[0028] Figure 3 This is the antibacterial colony characteristic diagram of the Bacillus subtilis LYZ1128 strain of this application against 11 common pathogens of alfalfa diseases and 1 pathogen of Oxytropis flavescentis disease;

[0029] Figure 4 This is a diagram showing the effects of protein decomposition, nitrogen fixation and potassium decomposition of Bacillus subtilis LYZ1128 of the present application;

[0030] Figure 5 This is a diagram showing the effect of Bacillus subtilis LYZ1128 on promoting root growth of alfalfa plants and preventing and controlling alfalfa Verticillium wilt;

[0031] Figure 6 This is a diagram showing the effect of Bacillus subtilis LYZ1128 on promoting the growth of alfalfa plants and preventing and controlling alfalfa Verticillium wilt;

[0032] Figure 7 This application is about the effect of Bacillus subtilis LYZ1128 on alfalfa plant height;

[0033] Figure 8 This application is about the effect of Bacillus subtilis LYZ1128 on the number of compound leaves of alfalfa;

[0034] Fig. 9 This application is about the effect of Bacillus subtilis LYZ1128 on the number of alfalfa branches;

[0035] Fig.10 This application is about the effect of Bacillus subtilis LYZ1128 on the diameter of the main stem of alfalfa;

[0036] Fig.11 For this application, the effect of Bacillus subtilis LYZ1128 on alfalfa root growth;

[0037] Fig.12 This application is about the effect of Bacillus subtilis LYZ1128 on the taproot diameter of alfalfa;

[0038] Fig.13This application is about the effect of Bacillus subtilis LYZ1128 on the fresh weight of alfalfa plant;

[0039] Fig.14 This application is about the effect of Bacillus subtilis LYZ1128 on the dry weight of alfalfa plant;

[0040] Fig.15 For this application, the effect of Bacillus subtilis LYZ1128 on the incidence of alfalfa;

[0041] Fig.16 This application is about the effect of Bacillus subtilis LYZ1128 on alfalfa disease index;

[0042] Fig.17 This is a diagram showing the control effect of Bacillus subtilis LYZ1128 on alfalfa in this application. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. In addition, it should be understood that the specific implementation methods described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0044] In this application, unless otherwise stated, directional words such as "upper" and "lower" generally refer to the upper and lower parts of the device in actual use or working state, specifically the drawing direction in the accompanying drawings; while "inner" and "outer" refer to the outline of the device. In addition, in the description of this application, the term "including" means "including but not limited to". The terms first, second, third, etc. are used only as labels and do not impose numerical requirements or establish an order.

[0045] In this application, "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0046] In the present application, "at least one" means one or more, and "plurality" means two or more. "One or several", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0047] Various embodiments of the present application may be presented 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 understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have 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., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0048] In a first aspect, an embodiment of the present invention provides Bacillus subtilis LYZ1128, and the deposit number of Bacillus subtilis LYZ1128 is CGMCC No.31719.

[0049] The present application separates a bacterium, Bacillus subtilis LYZ1128, from alfalfa stems. The bacterium is identified as Bacillus subtilis LYZ1128 by morphology combined with molecular biology, and is preserved with a preservation number of CGMCC No.31719. The strain has good antibacterial activity against 11 pathogens of 6 common alfalfa diseases and Verticillium dahliae (isolated from Oxytropis flavescentis plants) of the same genus but different species as Verticillium alfalfa. Potted plant experiments show that Bacillus subtilis LYZ1128 can promote the growth of alfalfa roots and plants, and can also significantly reduce the incidence of alfalfa Verticillium wilt, and has a good control effect on alfalfa Verticillium wilt, providing an important theoretical basis for the biological control of alfalfa Verticillium wilt.

[0050] In this application, Bacillus subtilis LYZ1128 is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, deposit number: CGMCC No.31719, deposit date: August 22, 2024, biological material (strain): LYZ1128, classification name: Bacillus subtilis.

[0051] In some embodiments, the 16S rDNA sequence of Bacillus subtilis LYZ1128 is as follows:

[0052] As shown in table SEQ ID No:1.

[0053] In some embodiments, the Bacillus subtilis LYZ1128 has a proteolytic effect.

[0054] In some embodiments, the Bacillus subtilis LYZ1128 has a nitrogen fixation function.

[0055] In some embodiments, the Bacillus subtilis LYZ1128 has a potassium-solubilizing effect.

[0056] In some embodiments, the Bacillus subtilis LYZ1128 is from Medicago sativa

[0057] L.) Stem.

[0058] It can be understood that the alfalfa stems are healthy alfalfa stems, and the alfalfa variety is WL343HQ; the collection location is: Liuxin Village, Minle County, Zhangye City, Gansu Province.

[0059] It can be understood that the alfalfa variety WL343HQ has good resistance to alfalfa Verticillium wilt ((V.alfalfae)LYZ0257).

[0060] In a second aspect, an embodiment of the present invention provides a biocontrol agent, wherein the biocontrol agent includes the Bacillus subtilis LYZ1128.

[0061] In some embodiments, the biocontrol agent further comprises auxiliary materials, wherein the auxiliary materials include sterile water, LB

[0062] At least one of the culture medium.

[0063] Furthermore, the number of viable bacteria of Bacillus subtilis LYZ1128 in the biocontrol agent is 10 5 CFU / mL~10 10 CFU / mL, for example, can be 10 5CFU / 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 bacterial count, there is a good biocontrol effect.

[0064] In a third aspect, an embodiment of the present invention provides an application of the biocontrol agent in preventing and controlling alfalfa diseases, wherein the alfalfa diseases are one or more of alfalfa verticillium wilt, alfalfa root rot, alfalfa stem spot mildew, alfalfa anthracnose, alfalfa stolonifer leaf spot and alfalfa Cangzhou leaf blight.

[0065] Furthermore, the pathogen of alfalfa Verticillium wilt is V.alfalfae LYZ0257; the pathogen of alfalfa root rot is one or more of Paraphoma radicina LYZ0187, Fusarium oxysporum LYZ0357, Fusarium solani LYZ0358, Paraphoma achrysanthemicola LYZ0368, Plectosphaerella cucumerina LYZ0547 and Fusarium tricinctum LYZ0551; the pathogen of alfalfa stem spot is Phoma medicaginis LYZ0429; the pathogen of alfalfa anthracnose is Colletotrichum spp. americae-borealis)LYZ0544; the pathogen of alfalfa leaf spot is Stemphylium botryosum LYZ0550; the pathogen of alfalfa leaf blight is Slafractonia leguminicola LYZ0582.

[0066] In a fourth aspect, an embodiment of the present invention provides the use of the biocontrol agent in promoting the growth of alfalfa.

[0067] Furthermore, the biocontrol agent promotes the growth of alfalfa roots.

[0068] Furthermore, the growth of alfalfa root system includes an increase in taproot length and taproot diameter.

[0069] Furthermore, the biocontrol agent promotes the growth of alfalfa plants.

[0070] Furthermore, the growth of alfalfa plants includes an increase in alfalfa plant height, an increase in the number of compound leaves, an increase in the diameter of the main stem, an increase in the fresh weight of a single plant, and an increase in the dry weight of a single plant.

[0071] In a fifth aspect, an embodiment of the present invention provides the use of the biocontrol agent in preventing and controlling diseases of Oxytropis tinctoria, wherein the disease of Oxytropis tinctoria is Verticillium wilt.

[0072] Furthermore, the pathogen of the Verticillium wilt disease of Oxytropis fulva is Verticillium dahliae LYZ0382.

[0073] In a sixth aspect, an embodiment of the present invention provides an anti-disease agent, wherein the anti-disease agent comprises the Bacillus subtilis LYZ1128.

[0074] Further, the disease resistance agent is caused by V.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 solani LYZ0358, Paraphoma chrysanthemicola LYZ0368, Phoma medicaginis LYZ0429, Colletotrichum americae-borealis LYZ0544, Plectosphaerella cucumerina LYZ0547, Stemphylium botryosum LYZ0550, Fusarium The invention is a disease-resistant agent for alfalfa diseases caused by tricinctum (LYZ0551) or Cangzhou leaf blight (Slafractonia leguminicola) LYZ0582.

[0075] Furthermore, the disease-resistant agent is a disease-resistant agent for the Oxytropis flavonoides disease caused by Verticillium dahliae LYZ0382.

[0076] Test Example 1

[0077] Morphological identification:

[0078] For identification of culture characteristics, the Bacillus subtilis LYZ1128 of the present application was streaked onto LB medium to obtain a single colony, cultured at 28° C., and its colony characteristics were continuously observed.

[0079] Gram staining: Gram staining was performed after culturing the Bacillus subtilis LYZ1128 of the present application at 28°C for 18 hours. A small amount of Bacillus subtilis LYZ1128 was picked up with a loop and evenly smeared on a glass slide dripped with distilled water, and fixed by flame three times. After adding crystal violet, stain for 1 minute, rinse with water until colorless, then add iodine solution and stain for 1 minute, rinse with water until colorless. Add 95% alcohol, swing the glass slide, decolorize for 20-60 seconds, rinse with water and dry, then stain with safranin for 1 minute, rinse with water, dry, and observe its individual morphological characteristics with an oil immersion lens.

[0080] Morphological observation results showed that the colonies of strain LYZ1128 cultured at 28°C for 72 h on LB medium were white, round, wrinkled, and had irregular edges ( Figure 1 A), Gram staining + , and the bacteria were observed to be rod-shaped, about 2-3 μm long ( Figure 1 B).

[0081] Among them, LB medium: 10.0 g of tryptone, 5.0 g of yeast extract, 5.0 g of NaCl, and 1 L of distilled water.

[0082] Test Example 2

[0083] Molecular Biology Identification:

[0084] A single colony of Bacillus subtilis LYZ1128 was streaked and purified on beef extract peptone medium (NA), and 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 the DNA were determined using a microspectrophotometer, and then the qualified DNA samples were stored in a -20°C refrigerator for PCR amplification.

[0085] PCR amplification, primers used were commonly used primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′. The PCR system was: DNA template 5μL, 2×Tap PCR Master Mix 25μL, forward and reverse primers 1μL each, ddH2O18μL. PCR amplification conditions were: 95℃ pre-denaturation for 4min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, 35 cycles; 72℃ extension for 10min. PCR amplification products were detected by 1.0% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0086] The sequencing results were compared with the sequences in the EzBioCloud database, and then the phylogenetic tree was constructed using MEGA7.0 software to determine the taxonomic status of the strain. All sequences were connected to multiple gene fragments into multi-gene fragments using SequenceMan software. The support strength of each branch of the phylogenetic tree was evaluated by the bootstrap value of 1000 repetitions.

[0087] Among them, nutrient agar medium (NA): peptone 10.0g, beef extract 3.0g, sodium chloride 5.0g, agar 15.0g, distilled water 1L, and the pH value was adjusted to 7.0-7.2 with NaOH.

[0088] According to the constructed phylogenetic tree, it was found that the antagonistic bacteria LYZ1128 and Bacillus subtilis (Bacillus subtilis accession number MW228433.1) were clustered together with a bootstrap value of 99%, which was smaller than the bootstrap values ​​of other species, so it can be identified as Bacillus subtilis ( Figure 2

[0089] Based on the results of morphological and molecular biological identification, strain LYZ1128 was identified as Bacillus subtilis (B. subtilis).

[0090] The sequence of SEQ ID No: 1 above was submitted as an electronic sequence listing file that complies with the WIPOST.26 standard.

[0091] Test Example 3

[0092] Antibacterial activity test:

[0093] The plate confrontation method was used to determine the antibacterial effect of Bacillus subtilis LYZ1128. Each pathogen to be tested was activated and cultured on a PDA plate for 3-5 days, and a bacterial cake (5 mm in diameter) was made and inoculated in the center of the PDA medium plate. The activated Bacillus subtilis LYZ1128 bacterial solution in the LB liquid medium was smeared at 4 cm on the left and right of the center of the plate, and cultured in the dark at 28°C for 7 days (different pathogens have different growth rates, and the control group is measured when the culture dish is full). The pathogens were inoculated on the PDA medium plate as a blank control. Each treatment was repeated 3 times, and the experiment was repeated 3 times independently. When the mycelium of each pathogen in the blank control group was full of the plate, the diameter of the pathogenic fungus was measured, and the relative inhibition rate was calculated according to the following formula.

[0094] Relative inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100.

[0095] Potato Dextrose Agar (PDA): 200.0 g potato, 20.0 g glucose, 20.0 g agar, 1 L distilled water;

[0096] The pathogens to be tested were fungal strains stored in the bacterial strain bank of the Grassland Microbiology Center of Lanzhou University, including V.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 (Fusarium tricinctum)LYZ0551, Cangzhou leaf blight (Slafractonia leguminicola)LYZ0582.

[0097] Figure 3A, control colony of Verticillium alfae; B, colony inhibited by LYZ1128 against Verticillium alfae 0257; C, control colony of P. radicina LYZ0187; D, colony inhibited by LYZ1128 against P. radicina LYZ0187; E, control colony of F. oxysporum LYZ0357; F, colony inhibited by LYZ1128 against F. oxysporum LYZ0357; G, colony inhibited by F. solani LYZ 0358; H, LYZ1128 inhibits colonies of F. solani LYZ0358; I, P. chrysanthemicola LYZ0368 control colonies; J, LYZ1128 inhibits colonies of P. chrysanthemicola LYZ0368; K, V. dahliae LYZ0382 control colonies; L, LYZ1128 inhibits colonies of V. dahliae LYZ0382; M, P. medicaginis L YZ0429 colony; N, LYZ1128 inhibited colony against P. medicaginis LYZ0429; O, C. americae-borealis LYZ0544 control colony; P, LYZ1128 inhibited colony against C. americae-borealis LYZ0544; Q, P. cucumerina LYZ0547 control colony; R, LYZ1128 inhibited colony against P. cucumerina LYZ0547; S, onion leaf blight Control colonies of S. botryosum LYZ0550; T, colonies inhibited by LYZ1128 against S. botryosum LYZ0550 of onion leaf blight; U, control colonies of F. tricinctum LYZ0551; V, colonies inhibited by LYZ1128 against F. tricinctum LYZ0551; W, control colonies of S. leguminicola LYZ0582; X, colonies inhibited by LYZ1128 against S. leguminicola LYZ0582 of onion leaf blight.

[0098] The results of the inhibition rate of Bacillus subtilis LYZ1128 against 11 alfalfa disease pathogens and 1 Oxytropis wilt pathogen are shown in Table 1.

[0099] Table 1 Inhibition rate of LYZ1128 against 11 common alfalfa pathogens and 1 pathogen of Verticillium wilt of Oxytropis erythrorhizon

[0100]

[0101] Note: Different letters in the same column indicate significant differences (P<0.05).

[0102] from Figure 3 As shown in Table 1, Bacillus subtilis LYZ1128 has an inhibitory effect on 11 alfalfa pathogens and 1 yellow flower bean Verticillium wilt pathogen, and the inhibition rate is significantly different (P<0.05). The inhibition rate of V.alfalfae LYZ0257 is 75.83% ( Figure 3 AB), P. radicina LYZ0187, the inhibition rate was 43.94% ( Figure 3 CD), Verticillium dahliae LYZ0382, the inhibition rate was 54.61% ( Figure 3 KL), F.oxysporum LYZ0357, the inhibition rate was 51.24% ( Figure 3 EF), Fusarium solani LYZ0358, the inhibition rate was 52.64% ( Figure 3 GH), P. chrysanthemicola LYZ0368, the inhibition rate was 37.89% ( Figure 3 IJ), P. medicaginis LYZ0429, the inhibition rate was 67.84% ( Figure 3 MN), North American anthrax (C.americae-borealis) LYZ054, the inhibition rate was 59.95% ( Figure 3 OP), P.cucumerina LYZ0547, the inhibition rate was 51.36% ( Figure 3 QR), S. botryosum LYZ0550, the inhibition rate was 85.59% ( Figure 3 ST), F.tricinctum LYZ0551, the inhibition rate was 42.17% ( Figure 3 UV), Cangzhou leaf blight (S.leguminicola) LYZ0582, the inhibition rate was 79.32% ( Figure 3 WX).

[0103] Test Example 4

[0104] Bacillus subtilis LYZ1128's ability to degrade protein, fix nitrogen and degrade potassium:

[0105] Nitrogen fixation ability test: Bacillus subtilis LYZ1128 was first activated on LB medium, and then inoculated into nitrogen-free medium, with 3 replicates for each treatment. Cultured at 28℃ for 7 days, if colonies appeared on the nitrogen-free medium, transferred to nitrogen-free medium again, cultured at 28℃ for 7 days, and transferred three times in succession. After three transfers, if colonies appeared on the nitrogen-free medium, it means that the strain has the ability to fix nitrogen.

[0106] Identification of protein degradation function: First, inoculate Bacillus subtilis LYZ1128 onto a skimmed milk powder plate and culture it at 37°C for 1-2 days. Observe the appearance of a transparent circle and measure the diameter of the transparent circle (D) and the diameter of the colony (d) at the same time. The protein degradation ability of the strain can be determined based on the ratio of the transparent circle diameter to the colony diameter (HC).

[0107] Identification of potassium-solubilizing function: First, Bacillus subtilis LYZ1128 was inoculated onto a silicate bacterial culture medium plate using the three-zone streak method, cultured at 37°C for 3-5 days, and observed whether the strain could produce smooth, transparent, oil-drop-shaped colonies that produced capsules.

[0108] like Figure 4 , Figure 4 A, protein-degrading colony characteristics of LYZ1128; B, nitrogen-fixing colony characteristics of LYZ1128; C, potassium-degrading colony characteristics of LYZ1128;

[0109] from Figure 4 It can be seen that Bacillus subtilis LYZ1128 inoculated on the surface of the protein-degrading culture medium produces a transparent circle, proving that the bacteria has the ability to degrade proteins (HC = 164.31) ( Figure 4 A); At the same time, the bacteria were inoculated in a nitrogen-fixing medium. After three transfers, colonies still grew, proving that the bacteria had a nitrogen-fixing effect ( Figure 4 B); when the bacteria is inoculated on the surface of potassium-dissolving medium, the strain can produce smooth and transparent oil-drop-shaped colonies that produce capsules ( Figure 4 C).

[0110] Test Example 5

[0111] Potted plant protection effect determination:

[0112] Preparation of spore suspension, biocontrol liquid: Bacillus subtilis LYZ1128 strain was inoculated into LB culture medium, cultured in a shake flask at 28°C and 200 r / min for 48 h, and then the culture medium was diluted with sterile water to a spore concentration of 10 8CFU / mL, set aside. Pathogen spore suspension: inject 10mL of sterile water on the surface of the PDA plate where the pathogen was cultured for 2 weeks, then gently scrape the surface of the colony with a sterilized slide to make the spores on the colony fall off, then pipette the spore liquid into a sterilized 50mL centrifuge tube, and then use a blood cell counting plate with the help of a microscope to adjust the spore concentration to 10 6 spores / mL for later use.

[0113] Germination and seedling raising: After surface disinfection, the alfalfa seeds of Xinmu No. 1 were placed on two layers of pathogen spore suspension or sterile water (control sterile water) soaked filter paper (sterilization), 45 seeds per dish, 5mL pathogen spore suspension or sterile water (control sterile water, treatment plus spore suspension) was injected into the dish every 24h for germination, and germination was promoted at 25℃ for 72h. Seeds with consistent germination were selected and planted in pots filled with sterile soil, 5 plants per pot. The seedlings were cultured in a greenhouse with a daytime temperature of 24±2℃, a nighttime temperature of 7±2℃, a humidity of 60%, 12h light, and 12h dark, and watered as needed.

[0114] There were 6 treatments in the experiment, each with 7 replicates (pots), namely (1) sterile water; (2) LB culture medium; (3) biocontrol strain LYZ1128; (4) sterile water + V.alfalfaeLYZ0257; (5) LB culture medium + V.alfalfaeLYZ0257 and (6) biocontrol strain LYZ1128 + V.alfalfaeLYZ0257.

[0115] Inoculation method: Pathogen inoculation used seed soaking method, biocontrol bacteria used potting method, seedlings germinated and soaked with sterile water (treatments 1 to 3) and seedlings germinated and soaked with V.alfalfaeLYZ0257 spore suspension (treatments 4 to 6), after the seedlings were transplanted into pots (inner diameter 14 cm) filled with sterilized soil (black charcoal soil) and grown for 7 days, the prepared biocontrol bacteria solution, water and LB culture medium were potted, 25 mL per pot and 5 mL per plant.

[0116] Index determination: 60 days after inoculation, plant height, number of leaves, number of branches, main stem diameter, root length, main root diameter, fresh weight, dry weight, number of diseased plants and severity were measured to calculate the incidence of alfalfa Verticillium wilt, disease index, and control effect. The classification standard for alfalfa Verticillium wilt and the severity of the disease are recorded in Table 2:

[0117] Incidence rate = (number of diseased plants / total number of plants surveyed) × 100%

[0118] Disease index = Σ(disease level × number of plants at that level) / (total number of plants surveyed × 5) × 100%

[0119] Control effect (%) = (disease index of control - disease index of treatment) / disease index of control × 100

[0120] Table 2 Severity classification of alfalfa Verticillium wilt

[0121]

[0122] Figure 5 From left to right in center A are alfalfa roots inoculated with biocontrol bacteria LYZ1128, LB culture medium, and sterile water. Figure 5 It can be seen that: compared with the control, the root length of alfalfa inoculated with only the biocontrol bacteria LYZ1128 increased significantly, and the biocontrol bacteria LYZ1128 is beneficial to the growth of alfalfa roots.

[0123] Figure 5 From left to right in middle B are alfalfa roots inoculated with biocontrol bacteria LYZ1128 + Verticillium lucerne LYZ0257, LB culture medium + Verticillium lucerne LYZ0257, and sterile water + Verticillium lucerne LYZ0257. Figure 5 It can be seen that after being infected by Verticillium oxysporum LYZ0257, compared with the control, the roots of alfalfa inoculated with the biocontrol bacteria LYZ1128 had no obvious symptoms of alfalfa Verticillium wilt, indicating that the biocontrol bacteria LYZ1128 can inhibit Verticillium oxysporum LYZ0257.

[0124] Figure 6 A in the middle (from right to left) shows alfalfa plants inoculated with sterile water, LB culture medium and biocontrol bacteria LYZ1128. Figure 6 It can be seen that: compared with the control, the height of alfalfa plants inoculated only with the biocontrol bacteria LYZ1128 increased significantly, and the biocontrol bacteria LYZ1128 is beneficial to the growth of alfalfa plants.

[0125] Figure 6 B (from right to left) shows alfalfa plants inoculated with sterile water + Verticillium alfalfa LYZ0257, LB culture medium + Verticillium alfalfa LYZ0257, and biocontrol bacteria LYZ1128 + Verticillium alfalfa LYZ0257. Figure 6 It can be seen that after being infected by alfalfa Verticillium wilt LYZ0257, the number of alfalfa plants inoculated with the biocontrol bacteria LYZ1128 that developed symptoms of alfalfa wilt was significantly reduced compared with the control. The alfalfa plants inoculated with sterile water and LB culture medium showed obvious symptoms of the disease, with yellowing of alfalfa leaves and typical "V"-shaped spots on the leaf tips.

[0126] from Figures 7 to 14 It can be seen that:

[0127] Bacillus subtilis (B. subtilis) LYZ1128 can significantly increase the plant height, number of compound leaves, main stem diameter, taproot length, taproot diameter, fresh weight per plant and dry weight per plant of alfalfa plants (P<0.05), while LB culture medium has no significant effect on alfalfa plants.

[0128] The heights of alfalfa plants treated with sterile water, LB medium and LYZ1128 were 36.5 cm, 36.8 cm and 40.2 cm, respectively. The number of compound leaves was 16, 22 and 31, respectively. The number of branches was 2, 2 and 2, respectively. The diameters of the main stems were 1.05 mm, 1.06 mm and 1.24 mm, the root lengths were 12.49 cm, 12.49 cm and 13.94 cm, respectively. The diameters of the main roots were 1.91 mm, respectively. , 1.95mm and 2.33mm, the fresh weight of a single plant was 1.45g, 1.46g and 1.85g, and the dry weight of a single plant was 0.39g, 0.41g and 0.41g, respectively. Compared with CK, the plant height increased by about 10% after LYZ1128 treatment; the number of compound leaves increased by 89%, the main stem diameter increased by 18%, the root length increased by 12%, the main root diameter increased by 22%, the fresh weight of a single plant increased by about 27%, and the dry weight of a single plant increased by about 6.3%. It can be seen that the biocontrol bacteria LYZ1128 has a significant effect on the growth indicators of alfalfa plants and alfalfa yield.

[0129] After infection with Verticillium alfalfa LYZ0257, the heights of alfalfa plants treated with sterile water, LB medium, and LYZ1128 were 29.3 cm, 29.3 cm, and 36.9 cm, respectively. Figure 7 ), the number of compound leaves is 18, 16 and 27 respectively ( Figure 8 ), the number of branches is about 2, 2 and 2 respectively ( Fig. 9 ), the main stem diameter is 1.07mm, 1.09mm and 1.19mm ( Fig.10 ), the root lengths are 10.66cm, 10.84cm and 12.5cm respectively ( Fig.11 ), the taproot diameters are 1.31mm, 1.34mm and 2.14mm respectively ( Fig.12 ), and the fresh weight of each plant was 0.92g, 0.90g and 1.51g respectively ( Fig.13 ), and the dry weight of each plant was 0.29g, 0.29g and 0.41g respectively ( Fig.14 ); Compared with CK, the plant height increased by about 26% after LYZ1128 treatment; the number of compound leaves increased by 48%, the main stem diameter increased by 11%, the root length increased by 17%, the main root diameter increased by 63%, the fresh weight of each plant increased by about 64%, and the dry weight of each plant increased by about 40%. It can be seen that after the alfalfa Verticillium ALFA LYZ0257 infects alfalfa plants, it has a significant impact on the alfalfa plants, but the biocontrol bacteria LYZ1128 can weaken the impact of the pathogen on alfalfa plants.

[0130] from Fig.15 Fig.16 and Fig.17 It can be seen that after the treatment with sterile water (CK), LB culture medium and Bacillus subtilis (B.subtilis) LYZ1128, the incidence of alfalfa Verticillium wilt was 91.4%, 88.6% and 34.29% respectively, and the disease index was 43.4, 42.9 and 9.1 respectively. The control effect of LB culture medium and Bacillus subtilis (B.subtilis) LYZ1128 on alfalfa Verticillium wilt was 1.3% and 78.95% respectively. It can be seen that Bacillus subtilis (B.subtilis) LYZ1128 can significantly reduce the incidence of alfalfa Verticillium wilt and has a good control effect on alfalfa Verticillium wilt.

[0131] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of the present application. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements and amendments to the present application. Such modifications, improvements and amendments are suggested in the present application, so such modifications, improvements and amendments still belong to the spirit and scope of the exemplary embodiments of the present application.

[0132] At the same time, the present application uses specific words to describe the embodiments of the present application. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of the present application. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, some features, structures or characteristics in one or more embodiments of the present application can be appropriately combined.

Claims

1. Bacillus subtilis LYZ1128, characterized in that The deposit number of the Bacillus subtilis LYZ1128 is CGMCC No.31719.

2. The Bacillus subtilis LYZ1128 according to claim 1, characterized in that The 16S rDNA sequence of the Bacillus subtilis LYZ1128 is shown in the sequence table as SEQ ID No:

1.

3. The Bacillus subtilis LYZ1128 according to claim 1, characterized in that The Bacillus subtilis LYZ1128 has a proteolytic effect; and / or The Bacillus subtilis LYZ1128 has a nitrogen-fixing function; and / or The Bacillus subtilis LYZ1128 has a potassium-dissolving effect.

4. A biocontrol agent, characterized in that: The biocontrol agent includes the Bacillus subtilis LYZ1128.

5. The biocontrol agent according to claim 4, characterized in that: The biocontrol agent further comprises auxiliary materials; and / or The number of viable bacteria of Bacillus subtilis LYZ1128 in the biocontrol agent is 10 5 CFU / mL~10 10 CFU / mL.

6. Use of the biocontrol agent according to claim 4 in preventing and controlling alfalfa diseases, wherein the alfalfa diseases are one or more of alfalfa Verticillium wilt, alfalfa root rot, alfalfa stem spot, alfalfa anthracnose, alfalfa stolonifer leaf spot and alfalfa Cangzhou leaf blight.

7. The use of the biocontrol agent according to claim 6 in preventing and controlling alfalfa diseases, characterized in that: The pathogen of alfalfa wilt is Verticillium alfalfae LYZ0257; the pathogens of alfalfa root rot are Paraphoma radicina LYZ0187, Fusarium oxysporum LYZ0357, Fusarium solani LYZ0358, Paraphoma chrysanthemicola LYZ0368, Plectosphaerella cucumerina LYZ0369, and LYZ0547 and one or more of Fusarium tricinctum LYZ0551; the pathogen of alfalfa stem spot is Phoma medicaginis LYZ0429; the pathogen of alfalfa anthracnose is Colletotrichum americae-borealis LYZ0544; the pathogen of alfalfa stalk spot is Stemphylium botryosum LYZ0550; the pathogen of alfalfa Cangzhou leaf blight is Slafractonia leguminicola LYZ0582.

8. Use of the biocontrol agent according to claim 4 in preventing and treating diseases of Oxytropis chinensis, wherein the disease of Oxytropis chinensis is Verticillium wilt of Oxytropis chinensis; and the pathogen of Verticillium wilt of Oxytropis chinensis is Verticillium dahliae LYZ0382.

9. Use of the biocontrol agent according to claim 4 in promoting the growth of alfalfa.

10. An anti-disease agent, characterized in that: The anti-disease agent includes the Bacillus subtilis LYZ1128; The disease-resistant agents are caused by Verticillium alfalfae LYZ0257, Paraphoma aradicina LYZ0187, Fusarium oxysporum LYZ0357, Fusarium solani LYZ0358, Paraphoma chrysanthemicola LYZ0368, Phoma medicaginis LYZ0429, Colletotrichumamericae-borealis LYZ0544, Plectosphaerella cucumerina LYZ0547, Stemphylium botryosum LYZ0550, Fusarium solani LYZ0358, Paraphoma chrysanthemicola LYZ0368, Phoma medicaginis LYZ0429, Colletotrichumamericae-borealis LYZ0544, Plectosphaerella cucumerina LYZ0547, Stemphylium botryosum LYZ0550, Fusarium tricinctum) LYZ0551 or alfalfa disease caused by Cangzhou leaf blight (Slafractonia leguminicola) LYZ0582; and / or The disease-resistant agent is a disease-resistant agent for the disease of Oxytropis flavonoides caused by Verticillium dahliae LYZ0382.

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