Bacillus amyloliquefaciens LYZ1125, biocontrol inoculant and application of biocontrol inoculant

Abstract

The invention provides bacillus amyloliquefaciens LYZ1125, a biocontrol microbial inoculum and application of the biocontrol microbial inoculum. The preservation number of the bacillus amyloliquefaciens LYZ1125 is CGMCC (China General Microbiological Culture Collection Center) No.31716, and the preservation number of the bacillus amyloliquefaciens LYZ1125 is CGMCC No.31716. The bacillus amyloliquefaciens LYZ1125 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 amyloliquefaciens LYZ1125 can promote the growth of alfalfa root systems and plants; the morbidity of the alfalfa verticillium wilt can be remarkably reduced, the good prevention and treatment effect on the alfalfa verticillium wilt is achieved, and an important theoretical basis is provided for biological prevention and treatment of the alfalfa verticillium wilt (V.alfalfae) LYZ0257).

Description

Technical Field

[0001] The present application relates to the technical field of plant disease prevention and control, and in particular to Bacillus amyloliquefaciens LYZ1125, biocontrol agents and applications thereof. Background Art

[0002] Alfalfa Verticillium alfalfae is a quarantined, destructive soil-borne disease. It has become an important fact that it has occurred on a large scale in the Hexi Corridor of my country. Previously, foreign studies have found that fumigating the diseased field soil twice with methyl bromide and chloropicrin has a good effect. The fumigation method was banned in my country in 2015 because methyl bromide can cause serious damage to the atmosphere.

[0003] 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 scale in the Hexi region. Nowadays, the use of bacterial biocontrol agents is the most common green prevention method. Bacillus is a Gram-positive bacterium that is ubiquitous in the environment and is usually associated with plants and the rhizosphere. However, there are few biocontrol strains for alfalfa diseases. So far, there are reports of Bacillus cereus for the prevention and control of alfalfa seedling damping-off, Streptomyces for the prevention and control of alfalfa seedling damping-off, and Bacillus amyloliquefaciens for the prevention and control of alfalfa anthracnose. It is urgent to find biocontrol strains for the biological control of alfalfa wilt. Summary of the invention

[0004] In view of this, the purpose of the present application is to provide Bacillus amyloliquefaciens LYZ1125, 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.

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

[0006] The embodiment of the present invention provides Bacillus amyloliquefaciens LYZ1125, and the deposit number of Bacillus amyloliquefaciens LYZ1125 is CGMCC No.31716.

[0007] In some embodiments, the 16S rDNA sequence of the Bacillus amyloliquefaciens LYZ1125 is shown in SEQ ID No: 1 in the sequence listing.

[0008] In some embodiments, the Bacillus amyloliquefaciens LYZ1125 has a proteolytic effect; and / or

[0009] The Bacillus amyloliquefaciens LYZ1125 has a nitrogen-fixing function; and / or

[0010] The Bacillus amyloliquefaciens LYZ1125 has a potassium-dissolving effect.

[0011] Accordingly, an embodiment of the present invention provides a biocontrol agent, which includes the Bacillus amyloliquefaciens LYZ1125.

[0012] In some embodiments, the biocontrol agent further comprises an auxiliary material; and / or

[0013] The number of viable bacteria of Bacillus amyloliquefaciens LYZ1125 in the biocontrol agent is 10 5 CFU / mL~10 10 CFU / mL.

[0014] 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.

[0015] In some embodiments, 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.

[0016] Accordingly, 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.

[0017] In some embodiments, the pathogen of Verticillium wilt of Oxytropis flavonoids is Verticillium dahliae LYZ0382.

[0018] Accordingly, an embodiment of the present invention provides the use of the biocontrol agent in promoting the growth of alfalfa.

[0019] Accordingly, an embodiment of the present invention provides an anti-disease agent, which includes the Bacillus amyloliquefaciens LYZ1125.

[0020] In some implementations, the disease-resistant 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

[0021] The disease-resistant agent is a disease-resistant agent for the disease of Oxytropis flavonoides caused by Verticillium dahliae LYZ0382. The present application isolates a bacterium, Bacillus amyloliquefaciens LYZ1125, from alfalfa stems. The bacterium is identified as Bacillus amyloliquefaciens LYZ1125 by morphology combined with molecular biology, and is preserved with a preservation number of CGMCC No.31716. The strain has good antibacterial activity against 11 pathogens of 6 common alfalfa diseases and Verticillium dahliae (isolated from Oxytropis alba plants) of the same genus but different species as Verticillium alfalfa. Potted plant experiments show that Bacillus amyloliquefaciens LYZ1125 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 ((V.alfalfae)LYZ0257). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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:

[0023] Figure 1 The colony characteristics and Gram-stained spore characteristics of the Bacillus amyloliquefaciens LYZ1125 strain grown on the surface of LB medium for 72 hours in this application;

[0024] Figure 2 Phylogenetic tree of Bacillus amyloliquefaciens LYZ1125 strain and related strains of this application;

[0025] Figure 3 This is the antibacterial colony characteristic diagram of the Bacillus amyloliquefaciens LYZ1125 strain of this application against 11 common pathogens of alfalfa diseases and 1 pathogen of Oxytropis flavescentis disease;

[0026] Figure 4 This is a diagram showing the effects of protein decomposition, nitrogen fixation and potassium decomposition of Bacillus amyloliquefaciens LYZ1125 of the present application;

[0027] Figure 5 This is a diagram showing the effect of Bacillus amyloliquefaciens LYZ1125 on promoting root growth of alfalfa plants and preventing and controlling alfalfa Verticillium wilt;

[0028] Figure 6 This is a diagram showing the effect of Bacillus amyloliquefaciens LYZ1125 on promoting the growth of alfalfa plants and preventing and controlling alfalfa Verticillium wilt;

[0029] Figure 7This application is about the effect of Bacillus amyloliquefaciens LYZ1125 on alfalfa plant height;

[0030] Figure 8 This application is about the effect of Bacillus amyloliquefaciens LYZ1125 on the number of compound leaves of alfalfa;

[0031] Fig. 9 This application is about the effect of Bacillus amyloliquefaciens LYZ1125 on the number of alfalfa branches;

[0032] Fig.10 This application is about the effect of Bacillus amyloliquefaciens LYZ1125 on the diameter of the main stem of alfalfa;

[0033] Fig.11 This application is about the effect of Bacillus amyloliquefaciens LYZ1125 on alfalfa root growth;

[0034] Fig.12 This application is about the effect of Bacillus amyloliquefaciens LYZ1125 on the taproot diameter of alfalfa;

[0035] Fig.13 This application is about the effect of Bacillus amyloliquefaciens LYZ1125 on the fresh weight of alfalfa plant;

[0036] Fig.14 This application is about the effect of Bacillus amyloliquefaciens LYZ1125 on the dry weight of alfalfa plant;

[0037] Fig.15 For this application, the effect of Bacillus amyloliquefaciens LYZ1125 on the incidence of alfalfa;

[0038] Fig.16 This application is about the effect of Bacillus amyloliquefaciens LYZ1125 on alfalfa disease index;

[0039] Fig.17 This is a diagram showing the control effect of Bacillus amyloliquefaciens LYZ1125 on alfalfa in this application. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In a first aspect, an embodiment of the present invention provides Bacillus amyloliquefaciens LYZ1125, and the deposit number of Bacillus amyloliquefaciens LYZ1125 is CGMCC No.31716.

[0046] The present application isolates a bacterium, Bacillus amyloliquefaciens LYZ1125, from alfalfa stems. The bacterium is identified as Bacillus amyloliquefaciens LYZ1125 by morphology combined with molecular biology, and is preserved with a preservation number of CGMCC No.31716. The strain has good antibacterial activity against 11 pathogens of 6 common alfalfa diseases and Verticillium dahliae (isolated from Oxytropis flavosum plants) of the same genus but different species as Verticillium alfalfa. Potted plant experiments show that Bacillus amyloliquefaciens LYZ1125 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.

[0047] In this application, Bacillus amyloliquefaciens LYZ1125 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.31716, deposit date: August 22, 2024, biological material (strain): LYZ1125, classification name: Bacillus amyloliquefaciens.

[0048] In some embodiments, the 16S rDNA sequence of the Bacillus amyloliquefaciens LYZ1125 is shown in SEQ ID No: 1 in the sequence listing.

[0049] In some embodiments, the Bacillus amyloliquefaciens LYZ1125 has a proteolytic effect.

[0050] In some embodiments, the Bacillus amyloliquefaciens LYZ1125 has a nitrogen-fixing function.

[0051] In some embodiments, the Bacillus amyloliquefaciens LYZ1125 has a potassium-solubilizing effect.

[0052] In some embodiments, the Bacillus amyloliquefaciens LYZ1125 is from Medicago sativa

[0053] L.) Stem.

[0054] 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.

[0055] It can be understood that the alfalfa variety WL343HQ has good resistance to alfalfa Verticillium wilt ((V.alfalfae)LYZ0257).

[0056] In a second aspect, an embodiment of the present invention provides a biocontrol agent, wherein the biocontrol agent includes the Bacillus amyloliquefaciens LYZ1125.

[0057] In some embodiments, the biocontrol agent further comprises auxiliary materials, wherein the auxiliary materials include sterile water, LB

[0058] At least one of the culture medium.

[0059] Furthermore, the number of viable bacteria of Bacillus amyloliquefaciens LYZ1125 in the biocontrol agent is 10 5 CFU / mL~10 10 CFU / mL, for example, 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 bacterial count, there is a good biocontrol effect.

[0060] 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.

[0061] 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.

[0062] In a fourth aspect, an embodiment of the present invention provides the use of the biocontrol agent in promoting the growth of alfalfa.

[0063] Furthermore, the biocontrol agent promotes the growth of alfalfa roots.

[0064] Furthermore, the growth of alfalfa root system includes an increase in taproot length and taproot diameter.

[0065] Furthermore, the biocontrol agent promotes the growth of alfalfa plants.

[0066] 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 number of branches, 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.

[0067] 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.

[0068] Furthermore, the pathogen of the Verticillium wilt disease of Oxytropis fulva is Verticillium dahliae LYZ0382.

[0069] In a sixth aspect, an embodiment of the present invention provides an anti-disease agent, wherein the anti-disease agent comprises the Bacillus amyloliquefaciens LYZ1125.

[0070] 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 lysozyme tricinctum (LYZ0551) or lysozyme leaf blight (Slafractonia leguminicola) (LYZ0582).

[0071] Furthermore, the disease-resistant agent is a disease-resistant agent for the Oxytropis flavonoides disease caused by Verticillium dahliae LYZ0382.

[0072] Test Example 1

[0073] Morphological identification:

[0074] For identification of culture characteristics, the Bacillus amyloliquefaciens LYZ1125 of the present application was streaked with a single colony on LB medium, cultured at 28° C., and its colony characteristics were continuously observed.

[0075] Gram staining: Gram staining was performed after culturing the Bacillus amyloliquefaciens LYZ1125 of the present application at 28°C for 18 hours. A small amount of Bacillus amyloliquefaciens LYZ1125 was picked up with a bacterial 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.

[0076] Morphological observation results showed that the colonies of strain LYZ1125 cultured at 28°C for 72 h on LB medium were white, round, smooth, and had neat edges ( Figure 1 A), Gram stain G+, and the bacteria were observed to be rod-shaped, about 2-4 μm long ( Figure 1 B).

[0077] 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.

[0078] Test Example 2

[0079] Molecular Biology Identification:

[0080] A single colony of Bacillus amyloliquefaciens LYZ1125 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] According to the constructed phylogenetic tree, we found that the antagonist LYZ1125 and Bacillus amyloliquefaciens (Accession No. JN700127.1) clustered together with a bootstrap value of 99%, which was smaller than that of other species, so it can be identified as Bacillus amyloliquefaciens ( Figure 2

[0085] Based on the results of morphological and molecular biological identification, strain LYZ1125 was identified as Bacillus amyloliquefaciens.

[0086] The above sequence ID No: 1 was also submitted with an electronic sequence listing file that complies with the WIPOST.26 standard.

[0087] Test Example 3

[0088] Antibacterial activity test:

[0089] The plate confrontation method was used to determine the antibacterial effect of Bacillus amyloliquefaciens LYZ1125. 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 amyloliquefaciens LYZ1125 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 was measured when the culture dish was 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.

[0090] Relative inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100.

[0091] Potato Dextrose Agar (PDA): 200.0 g potato, 20.0 g glucose, 20.0 g agar, 1 L distilled water;

[0092] 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.

[0093] Figure 3A, control colony of V.alfalfae; B, colony inhibited by LYZ1125 against V.alfalfae 0257; C, control colony of P.radicina LYZ0187; D, colony inhibited by LYZ1125 against P.radicina LYZ0187; E, control colony of F.oxysporum LYZ0357; F, colony inhibited by LYZ1125 against F.oxysporum LYZ0357; G, colony inhibited by F.solani LYZ 0358; H, LYZ1125 inhibits the colony of Fusarium solani LYZ0358; I, P. chrysanthemicola LYZ0368 control colony; J, LYZ1125 inhibits the colony of P. chrysanthemicola LYZ0368; K, V. dahliae LYZ0382 control colony; L, LYZ1125 inhibits the colony of V. dahliae LYZ0382; M, P. medicaginis L YZ0429 colony; N, LYZ1125 inhibited colony against P. medicaginis LYZ0429; O, C. americae-borealis LYZ0544 control colony; P, LYZ1125 inhibited colony against C. americae-borealis LYZ0544; Q, P. cucumerina LYZ0547 control colony; R, LYZ1125 inhibited colony against P. cucumerina LYZ0547; S, onion leaf blight Control colonies of S. botryosum LYZ0550; T, colonies inhibited by LYZ1125 against S. botryosum LYZ0550 of onion leaf blight; U, control colonies of F. tricinctum LYZ0551; V, colonies inhibited by LYZ1125 against F. tricinctum LYZ0551; W, control colonies of S. leguminicola LYZ0582; X, colonies inhibited by LYZ1125 against S. leguminicola LYZ0582 of onion leaf blight.

[0094] The inhibition rates of Bacillus amyloliquefaciens LYZ1125 against 11 alfalfa pathogens and 1 Oxytropis wilt pathogen are shown in Table 1.

[0095] Table 1 Inhibition rate of LYZ1125 against 11 common alfalfa pathogens and 1 pathogen of Verticillium wilt of Oxytropis erythrorhizon

[0096]

[0097]

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

[0099] from Figure 3 As shown in Table 1, Bacillus amyloliquefaciens LYZ1125 has an inhibitory effect on 11 common alfalfa pathogens and 1 yellow bean wilt pathogen. The inhibition rate of Colletotrichum americae-borealis LYZ0544 and Plectosphaerella cucumerina LYZ0547 is not significantly different, and the inhibition rate of Plectosphaerella cucumerina LYZ0547 and Slafractonia leguminicola LYZ0582 is not significantly different. The other pathogens are significantly different, specifically: the inhibition rate is significantly different (P<0.05). The inhibition rate of V.alfalfae LYZ0257 is 80.56% ( Figure 3 AB), P. radicina LYZ0187, the inhibition rate was 59.91% ( Figure 3 CD), Verticillium dahliae LYZ0382, the inhibition rate was 82.18% ( Figure 3 KL), F.oxysporum LYZ0357, the inhibition rate was 57.85% ( Figure 3 EF), Fusarium solani LYZ0358, the inhibition rate was 69.25% ( Figure 3 GH), P. chrysanthemicola LYZ0368, the inhibition rate was 55.65% ( Figure 3 IJ), P. medicaginis LYZ0429, the inhibition rate was 82.73% ( Figure 3 MN), North American anthrax (C.americae-borealis) LYZ054, the inhibition rate was 75.59% ( Figure 3 OP), P.cucumerina LYZ0547, the inhibition rate was 76.02% ( Figure 3QR), S. botryosum LYZ0550, the inhibition rate was 88.04% ( Figure 3 ST), F.tricinctum LYZ0551, the inhibition rate was 64.87% ( Figure 3 UV), Cangzhou leaf blight (S.leguminicola) LYZ0582, the inhibition rate was 76.2% ( Figure 3 WX).

[0100] Test Example 4

[0101] Protein-solubilizing, nitrogen-fixing and potassium-solubilizing abilities of Bacillus amyloliquefaciens LYZ1125:

[0102] Nitrogen fixation ability test: Bacillus amyloliquefaciens LYZ1125 was first activated on LB medium, and then inoculated into nitrogen-free medium, with 3 replicates for each treatment. Cultured at 28°C for 7 days, if colonies appeared on the nitrogen-free medium, transferred to nitrogen-free medium again, cultured at 28°C 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.

[0103] Identification of protein decomposition function: First, inoculate Bacillus amyloliquefaciens LYZ1125 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 decomposition ability of each strain can be determined based on the ratio of the transparent circle diameter to the colony diameter (HC).

[0104] Identification of potassium-solubilizing function: First, inoculate Bacillus amyloliquefaciens LYZ1125 onto a silicate bacterial culture medium plate using the three-zone streak method, culture at 37°C for 3-5 days, and observe whether the strain can produce smooth, transparent, oil-drop-shaped colonies that produce capsules.

[0105] like Figure 4 , Figure 4 A, protein-degrading colony characteristics of LYZ1125; B, nitrogen-fixing colony characteristics of LYZ1125; C, potassium-degrading colony characteristics of LYZ1125;

[0106] from Figure 4 It can be seen that a transparent circle is produced when Bacillus amyloliquefaciens LYZ1125 is inoculated on the surface of the protein-degrading culture medium, proving that the bacterium has the ability to degrade proteins (HC=6.1) ( 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, transparent, oil-drop-shaped colonies that produce capsules ( Figure 4 C).

[0107] Test Example 5

[0108] Potted plant protection effect determination:

[0109] Preparation of spore suspension, biocontrol liquid: Bacillus amyloliquefaciens LYZ1125 strain was inoculated into LB culture medium, cultured in a shaking 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 8 CFU / 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.

[0110] 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.

[0111] There were 6 treatments in the experiment, each with 7 replicates (pots), namely (1) sterile water; (2) LB culture medium; (3) biocontrol strain LYZ1125; (4) sterile water + V.alfalfaeLYZ0257; (5) LB culture medium + V.alfalfaeLYZ0257 and (6) biocontrol strain LYZ1125 + V.alfalfaeLYZ0257.

[0112] 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.

[0113] 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:

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

[0115] Disease index = Σ(disease level × number of plants at that 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 Severity classification of alfalfa Verticillium wilt

[0118]

[0119] Figure 5 From left to right in center A are alfalfa roots inoculated with biocontrol bacteria LYZ1125, LB culture solution, and sterile water. Figure 5 It can be seen that compared with the control, the root length of alfalfa inoculated with the biocontrol bacteria LYZ1125 increased significantly, and the biocontrol bacteria LYZ1125 is beneficial to the growth of alfalfa roots.

[0120] Figure 5 From left to right in middle B are alfalfa roots inoculated with biocontrol bacteria LYZ1125 + 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 LYZ1125 had no obvious symptoms of alfalfa Verticillium wilt, indicating that the biocontrol bacteria LYZ1125 can inhibit Verticillium oxysporum LYZ0257.

[0121] Figure 6 A in the middle is alfalfa plant inoculated with sterile water, LB culture medium and biocontrol bacteria LYZ1125. Figure 6 It can be seen that compared with the control, the height of alfalfa plants inoculated with only the biocontrol bacteria LYZ1125 increased significantly, and the biocontrol bacteria LYZ1125 is beneficial to the growth of alfalfa plants.

[0122] Figure 6 B in the middle is alfalfa plants inoculated with sterile water + Verticillium alfalfa LYZ0257, LB culture medium + Verticillium alfalfa LYZ0257, and biocontrol bacteria LYZ1125 + Verticillium alfalfa LYZ0257. Figure 6It can be seen that after being infected by alfalfa Verticillium wilt LYZ0257, the number of alfalfa plants inoculated with the biocontrol bacteria LYZ1125 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.

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

[0124] Bacillus amyloliquefaciens LYZ1125 can significantly increase the plant height, number of compound leaves, main stem diameter, taproot length, taproot diameter, fresh weight and dry weight of alfalfa plants (P<0.05), while LB culture medium has no significant effect on alfalfa plants.

[0125] The heights of alfalfa plants treated with sterile water, LB medium and LYZ1125 were 36.5 cm, 36.7 cm and 39.3 cm, respectively. The number of compound leaves was 16, 22 and 29, 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 1.17 mm, respectively. The root lengths were 12.49 cm, 12.49 cm and 14.21 cm, respectively. The main root diameters were 1.91 m, respectively. m, 1.95mm and 2.23mm, the fresh weight of a single plant was 1.45g, 1.46g and 1.91g, and the dry weight of a single plant was 0.39g, 0.41g and 0.46g, respectively. Compared with CK, the plant height increased by about 8% after LYZ1125 treatment; the number of compound leaves increased by 74%, the main stem diameter increased by 11%, the root length increased by 14%, the main root diameter increased by 17%, the fresh weight of a single plant increased by about 32%, and the dry weight of a single plant increased by about 19%. It can be seen that the biocontrol bacteria LYZ1125 has a significant effect on the growth indicators of alfalfa plants and alfalfa yield.

[0126] After infection with Verticillium alfalfa LYZ0257, the heights of alfalfa plants treated with sterile water, LB culture medium, and LYZ1125 were 29.3 cm, 29.3 cm, and 38.0 cm, respectively. Figure 7 ), the number of compound leaves is 18, 16 and 29 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 1.29mm ( Fig.10 ), the root lengths are 10.66cm, 10.84cm and 13.43cm respectively ( Fig.11 ), the taproot diameters are 1.31mm, 1.34mm and 2.09mm respectively ( Fig.12 ), and the fresh weight of each plant was 0.92g, 0.90g and 1.52g respectively ( Fig.13), and the dry weight of each plant was 0.29g, 0.29g and 0.40g respectively ( Fig.14 ); Compared with sterile water, the plant height increased by about 30% after LYZ1125 treatment; the number of compound leaves increased by 63%, the main stem diameter increased by 21%, the root length increased by 26%, the main root diameter increased by 59%, the fresh weight of each plant increased by about 64%, and the dry weight of each plant increased by about 39%. 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 LYZ1125 can weaken the impact of the pathogen on the alfalfa plants.

[0127] 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 amyloliquefaciens (B. amyloliquefaciens) LYZ1125, the incidence of alfalfa Verticillium wilt was 91.4%, 88.6% and 34.3% respectively, and the disease index was 43.4, 42.9 and 7.4 respectively. The control effect of LB culture medium and Bacillus amyloliquefaciens (B. amyloliquefaciens) LYZ1125 on alfalfa Verticillium wilt was 1.3% and 82.9% respectively. It can be seen that Bacillus amyloliquefaciens (B. amyloliquefaciens) LYZ1125 can significantly reduce the incidence of alfalfa Verticillium wilt and has a good control effect on alfalfa Verticillium wilt.

[0128] 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.

[0129] 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 amyloliquefaciens LYZ1125, characterized in that The deposit number of the Bacillus amyloliquefaciens LYZ1125 is CGMCC No.31716.

2. The Bacillus amyloliquefaciens LYZ1125 according to claim 1, characterized in that The 16S rDNA sequence of the Bacillus amyloliquefaciens LYZ1125 is shown in the sequence table as SEQ ID No:

1.

3. The Bacillus amyloliquefaciens LYZ1125 according to claim 1, characterized in that The Bacillus amyloliquefaciens LYZ1125 has a protein-degrading effect; and / or The Bacillus amyloliquefaciens LYZ1125 has a nitrogen-fixing function; and / or The Bacillus amyloliquefaciens LYZ1125 has a potassium-dissolving effect.

4. A biocontrol agent, characterized in that: The biocontrol agent includes the Bacillus amyloliquefaciens LYZ1125.

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 amyloliquefaciens LYZ1125 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 amyloliquefaciens LYZ1125; 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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