Bacillus amyloliquefaciens LYZ1125, biocontrol agent and application thereof

Abstract

The present application provides Bacillus amyloliquefaciens LYZ1125, a biocontrol agent, and applications thereof. The deposit number of the Bacillus amyloliquefaciens LYZ1125 is CGMCC No. 31716. The Bacillus amyloliquefaciens LYZ1125 of the present application has good antibacterial activity against 11 pathogens of 6 common alfalfa diseases and Verticillium dahliae (isolated from Oxytropis flavescentis plants), which is a different species from the same genus as Verticillium alfalfa. Potted plant experiments have shown that Bacillus amyloliquefaciens LYZ1125 can promote the growth of alfalfa roots and plants, and can also significantly reduce the incidence of alfalfa Verticillium wilt. It 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).

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 wilt (Verticillium alfalfae) is a quarantine-restricted, devastating soil-borne disease, with widespread occurrence in my country's Hexi Corridor. Previous studies abroad have shown that fumigating diseased fields twice with methyl bromide and chloropicrin is effective. However, fumigation was banned in my country in 2015 due to the severe damage methyl bromide can cause to the atmosphere.

[0003] Given the shortcomings of the above control methods, it is extremely important to find a green and environmentally friendly control method that can be used to control alfalfa verticillium wilt on a large scale in the Hexi region. Currently, the most common green control method is the use of bacterial biocontrol agents. Bacillus is a Gram-positive bacterium that is ubiquitous in the environment and is typically associated with plants and the rhizosphere. However, there are relatively few biocontrol strains for alfalfa diseases. To date, the reported strains include Bacillus cereus for controlling alfalfa seedling damping-off, Streptomyces for controlling alfalfa seedling damping-off, and Bacillus amyloliquefaciens for controlling alfalfa anthracnose. Therefore, the search for biocontrol strains for the biological control of alfalfa verticillium wilt is urgent. 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 wilt (Verticillium alfalfae) has been found in the prior art.

[0005] The technical solutions adopted by this application to solve the above technical problems are:

[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 the use 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, alfalfa anthracnose, alfalfa sclerotinia 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 chrysanthemicola LYZ0368, Plectosphaerella cucumerina LYZ0547, and Fusarium tricinctum LYZ0551; the pathogen of alfalfa leaf 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 flavescentis, wherein the disease of Oxytropis flavescentis is Verticillium wilt.

[0017] In some embodiments, the pathogen of Oxytropis flavescentis Verticillium wilt 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 embodiments, 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 Disease resistance agents for alfalfa diseases caused by lysozyme tricinctum (LYZ0551) or Cangzhou leaf blight (Slafractonia leguminicola) (LYZ0582); and / or

[0021] The disease-resistant agent is a disease-resistant agent for the oxtera bean disease caused by Verticillium dahliae LYZ0382. The present application isolated a bacterium, Bacillus amyloliquefaciens LYZ1125, from alfalfa stems. The bacterium was identified as Bacillus amyloliquefaciens LYZ1125 based on morphology and molecular biology, and was deposited with the deposit number CGMCC No. 31716. The strain showed good antibacterial activity against 11 pathogens of six common alfalfa diseases, as well as Verticillium dahliae (isolated from Oxytropis flavescentis plants), a different species from the same genus as Verticillium alfalfa. Potted experiments showed that Bacillus amyloliquefaciens LYZ1125 can promote the growth of alfalfa roots and plants, and can also significantly reduce the incidence of alfalfa Verticillium wilt. It has a good preventive and 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 following briefly introduces the drawings of the embodiments. Obviously, the drawings described below only relate to some embodiments of the present application and are not intended to limit the present application.

[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 in this application;

[0025] Figure 3 This is the antibacterial colony characteristic diagram of the Bacillus amyloliquefaciens LYZ1125 strain in 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 this application;

[0027] Figure 5 This is a diagram showing the effects 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 studies the effect of Bacillus amyloliquefaciens LYZ1125 on alfalfa plant height;

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

[0031] Figure 9 This application studies the effect of Bacillus amyloliquefaciens LYZ1125 on the number of alfalfa branches;

[0032] Figure 10 This application studies the effect of Bacillus amyloliquefaciens LYZ1125 on the diameter of the main stem of alfalfa;

[0033] Figure 11 This application studies the effect of Bacillus amyloliquefaciens LYZ1125 on alfalfa root growth;

[0034] Figure 12 This application studies the effect of Bacillus amyloliquefaciens LYZ1125 on the diameter of the main root of alfalfa;

[0035] Figure 13 This application studies the effect of Bacillus amyloliquefaciens LYZ1125 on the fresh weight of alfalfa plants;

[0036] Figure 14 This application studies the effect of Bacillus amyloliquefaciens LYZ1125 on the dry weight of alfalfa plants;

[0037] Figure 15 This application studies the effect of Bacillus amyloliquefaciens LYZ1125 on the incidence of alfalfa;

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

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

[0040] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only 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 making creative work are within the scope of protection of this application. In addition, it should be understood that the specific embodiments 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 indicated, directional terms such as "upper" and "lower" generally refer to the upper and lower positions of a device in actual use or operation, specifically in the drawing directions of the accompanying drawings; whereas "inner" and "outer" refer to the outline of the device. Furthermore, in the description of this application, the term "including" means "including but not limited to." Terms such as first, second, and third are used merely as designations and do not impose numerical requirements or establish a sequential order.

[0042] In this application, "and / or" describes the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can mean: 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 this 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, and c can be single or multiple.

[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 hard 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 numbers within the range. For example, the description of a range 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 applies 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, wherein the deposit number of Bacillus amyloliquefaciens LYZ1125 is CGMCC No.31716.

[0046] The present application describes a bacterial strain isolated from alfalfa stems, Bacillus amyloliquefaciens LYZ1125, which was identified as Bacillus amyloliquefaciens LYZ1125 based on morphology and molecular biology. The strain was deposited with the deposit number CGMCC No. 31716. The strain exhibits good antibacterial activity against 11 pathogens of six common alfalfa diseases, as well as Verticillium dahliae (isolated from Oxytropis flavescentis plants), a different species from Verticillium avium. Potted plant experiments indicate that Bacillus amyloliquefaciens LYZ1125 can promote root and plant growth of alfalfa, significantly reduce the incidence of alfalfa Verticillium wilt, and exhibit a good preventive and therapeutic effect against 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 of the China Culture Collection Administration (CGMCC), 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 time: 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 nitrogen fixation ability.

[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, which includes the Bacillus amyloliquefaciens LYZ1125.

[0057] In some embodiments, the biocontrol agent further comprises an auxiliary material, wherein the auxiliary material comprises 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, a better biocontrol effect is achieved.

[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, alfalfa anthracnose, alfalfa sclerotinia 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 chrysanthemicola LYZ0368, Plectosphaerella cucumerina LYZ0547 and Fusarium tricinctum LYZ0551; the pathogen of alfalfa leaf spot is Phoma medicaginis LYZ0429; the pathogen of alfalfa anthracnose is Colletotrichum fuscae. 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, embodiments of the present invention provide use of the biocontrol agent in promoting alfalfa growth.

[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 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 a use of the biocontrol agent in preventing and controlling diseases of Oxytropis flavescentis, wherein the disease of Oxytropis flavescentis is Verticillium wilt.

[0068] Furthermore, the pathogen of the yellow-flowered oxtroyan verticillium wilt disease is Verticillium dahliae LYZ0382.

[0069] In a sixth aspect, embodiments of the present invention provide an anti-disease agent, comprising the Bacillus amyloliquefaciens LYZ1125.

[0070] Furthermore, 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 relates to an agent for resisting alfalfa diseases caused by tricinctum) LYZ0551 or Cangzhou leaf blight (Slafractonia leguminicola) LYZ0582.

[0071] Furthermore, the disease resistance agent is an agent for resisting the disease of Oxytropis flavescentis 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 culture medium, cultured at 28° C., and its colony characteristics were continuously observed.

[0075] Gram staining: Culture the Bacillus amyloliquefaciens LYZ1125 strain described herein at 28°C for 18 hours and then perform Gram staining. Use a loop to pick a small amount of Bacillus amyloliquefaciens LYZ1125 and spread it evenly on a glass slide dripping with distilled water. Fix the strain by flame three times. Add crystal violet and 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 and shake the slide to decolorize for 20-60 seconds. Rinse with water, blot dry, and stain with safranin for 1 minute. Rinse with water, let dry, and observe individual morphological characteristics using an oil immersion lens.

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

[0077] 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). Then, a single colony was picked and the DNA concentration and quality were determined using the Ezup column-type bacterial genomic DNA extraction kit (B518255-0050) produced by Shanghai Biotechnology Co., Ltd. A microspectrophotometer was used to determine the DNA concentration and quality. The qualified DNA samples were then stored in a -20°C refrigerator for PCR amplification.

[0081] PCR amplification was performed using the commonly used primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′. The PCR system consisted of 5 μL of DNA template, 25 μL of 2×Tap PCR Master Mix, 1 μL each of forward and reverse primers, and 18 μL of ddH2O. PCR amplification conditions were: 95°C initial denaturation for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by extension at 72°C for 10 min. PCR products were verified by 1.0% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0082] To construct a phylogenetic tree, sequencing results were compared with sequences in the EzBioCloud database. A phylogenetic tree was then constructed using MEGA 7.0 software to determine the taxonomic status of the strains. Sequences were concatenated into multi-gene fragments using SequenceMan software. The strength of support for each branch of the phylogenetic tree was assessed using a bootstrap test with 1000 replicates.

[0083] Among them, nutrient agar (NA) medium: 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 antagonistic bacteria LYZ1125 and Bacillus amyloliquefaciens (accession number JN700127.1) clustered together with a self-bootstrapping value of 99%, which was smaller than that of other species. Therefore, 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 detection:

[0089] The plate standoff 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. 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 LB liquid medium was smeared 4 cm to 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 time it takes for the control group to fill the culture plate is used for determination). 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 hyphae of each pathogen in the blank control group filled 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 potatoes, 20.0 g glucose, 20.0 g agar, 1 L distilled water;

[0092] The pathogens to be tested were fungal strains deposited 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 colonies of Fusarium solani LYZ0358; I, control colonies of P. chrysanthemicola LYZ0368; J, LYZ1125 inhibits colonies of P. chrysanthemicola LYZ0368; K, control colonies of V. dahliae LYZ0382; L, LYZ1125 inhibits colonies of V. dahliae LYZ0382; M, P. medicaginis L YZ0429 colony; N, colony inhibited by LYZ1125 against P. medicaginis LYZ0429; O, control colony of C. americae-borealis LYZ0544; P, colony inhibited by LYZ1125 against C. americae-borealis LYZ0544; Q, control colony of P. cucumerina LYZ0547; R, colony inhibited by LYZ1125 against P. cucumerina LYZ0547; S, onion leaf blight Control colonies of S. botryosum LYZ0550; T, colonies inhibited by LYZ1125 against S. botryosum LYZ0550; 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.

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

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

[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. There is no significant difference in the inhibition rate between North American anthracnose (Colletotrichum americae-borealis) LYZ0544 and cucumber weaving spherical shell fungus (Plectosphaerella cucumerina) LYZ0547, and there is no significant difference in the inhibition rate between cucumber weaving spherical shell fungus (Plectosphaerella cucumerina) LYZ0547 and Cangzhou leaf blight (Slafractonia leguminicola) LYZ0582. There are significant differences between the other pathogens. 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), Fusarium oxysporum LYZ0357, the inhibition rate was 57.85% ( Figure 3 EF), Fusarium solani (F.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-solution, nitrogen-fixation and potassium-solution capabilities of Bacillus amyloliquefaciens LYZ1125:

[0102] Nitrogen-fixing ability test: First, activate Bacillus amyloliquefaciens LYZ1125 in LB medium and then inoculate it into nitrogen-free medium. Each treatment is replicated three times. Cultivate at 28°C for 7 days. If colonies appear on the nitrogen-free medium, transfer the strain to the nitrogen-free medium and incubate at 28°C for another 7 days. Repeat this transfer three times. If colonies appear on the nitrogen-free medium after each transfer, the strain has the ability to fix nitrogen.

[0103] Identification of protein degradation function: First, inoculate Bacillus amyloliquefaciens LYZ1125 onto a skim milk powder plate and culture at 37℃ for 1-2 days. Observe for the appearance of a transparent zone and simultaneously measure the diameter of the transparent zone (D) and the diameter of the colony (d). The protein degradation ability of each strain can be judged based on the ratio of the transparent zone diameter to the colony diameter (HC).

[0104] Identification of potassium-solubilizing function: First, Bacillus amyloliquefaciens LYZ1125 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.

[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 Bacillus amyloliquefaciens LYZ1125 inoculated on the surface of the protein-degrading culture medium produces a transparent circle, proving that the bacteria has the ability to degrade protein (HC = 6.1) ( Figure 4 A); At the same time, the bacteria were inoculated into a nitrogen-fixing culture 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 with capsules ( Figure 4 C).

[0107] Test Example 5

[0108] Potted plant efficacy determination:

[0109] Preparation of spore suspension, biocontrol solution: Bacillus amyloliquefaciens LYZ1125 strain was inoculated into LB culture medium, cultured in a shake flask at 28°C and 200 rpm 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 onto the surface of the PDA plate where the pathogens were cultured for 2 weeks, then gently scrape the surface of the colony with a sterile glass slide to remove the spores on the colony surface, and then pipette the spore solution into a sterile 50mL centrifuge tube. Then, use a hemocytometer to count the spores under a microscope and adjust the concentration to 10 6 spores / mL, set aside.

[0110] For germination and seedling cultivation, Xinmu No. 1 alfalfa seeds were surface-disinfected and placed on two layers of filter paper soaked in pathogen spore suspension or sterile water (sterile water for control). Each dish contained 45 seeds. Every 24 hours, 5 mL of pathogen spore suspension or sterile water (sterile water for control, spore suspension for treatment) was injected into the dish to accelerate germination. Germination was accelerated at 25°C for 72 hours. Seeds with consistent germination were selected and planted in pots filled with sterile soil, with 5 plants per pot. Seedlings were cultured in a greenhouse under conditions of 24 ± 2°C daytime temperature, 7 ± 2°C nighttime temperature, 60% humidity, 12 hours of light and 12 hours of darkness, and watered as needed.

[0111] Experimental treatments: The experiment mainly consists of 6 treatments, each with 7 (pot) replicates, 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: The pathogen was inoculated using the seed soaking method, and the biocontrol bacteria were inoculated using the pot rooting method. The seedlings (treatments 1 to 3) were germinated and soaked with sterile water and the seedlings (treatments 4 to 6) were germinated and soaked with V. alfalfae LYZ0257 spore suspension. 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 pot rooted, 25 mL per pot and 5 mL per plant.

[0113] Index measurement: 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 and disease index of alfalfa wilt, as well as the control effect. The grading standard for alfalfa 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 Alfalfa Verticillium wilt severity classification

[0118]

[0119] Figure 5 From left to right in center A are alfalfa roots inoculated with biocontrol bacteria LYZ1125, LB culture medium 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, the middle B shows the alfalfa roots inoculated with biocontrol bacteria LYZ1125 + Verticillium medicagogue LYZ0257, LB culture medium + Verticillium medicagogue LYZ0257, and sterile water + Verticillium medicagogue LYZ0257. Figure 5 It can be seen that after being infected by Verticillium oxysporum LYZ0257, the roots of alfalfa inoculated with the biocontrol bacteria LYZ1125 showed no obvious symptoms of alfalfa verticillium wilt compared with the control, 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 only with the biocontrol bacteria LYZ1125 increased significantly, and the biocontrol bacteria LYZ1125 is beneficial to the growth of alfalfa plants.

[0122] Figure 6 Figure B shows alfalfa plants inoculated with sterile water + Verticillium truncatum LYZ0257, LB culture medium + Verticillium truncatum LYZ0257, and biocontrol bacteria LYZ1125 + Verticillium truncatum LYZ0257. Figure 6It can be seen that after being infected by Verticillium oxysporum 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 cultured 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, main root length, main root 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 main stem diameters 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 mm, respectively. m, 1.95 mm, and 2.23 mm, with fresh weight per plant of 1.45 g, 1.46 g, and 1.91 g, respectively, and dry weight per plant of 0.39 g, 0.41 g, and 0.46 g, respectively. Compared with CK, LYZ1125 treatment increased plant height by approximately 8%, compound leaf number by 74%, main stem diameter by 11%, root length by 14%, taproot diameter by 17%, fresh weight per plant by approximately 32%, and dry weight per plant by approximately 19%. Therefore, the biocontrol fungus LYZ1125 significantly affected alfalfa plant growth indicators and yield.

[0126] After infection with Verticillium truncatum 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 are 18, 16 and 29 respectively ( Figure 8 ), the number of branches are approximately 2, 2 and 2 ( Figure 9 ), the main stem diameters are 1.07mm, 1.09mm and 1.29mm ( Figure 10 ), with root lengths of 10.66cm, 10.84cm, and 13.43cm ( Figure 11 ), the main root diameters were 1.31mm, 1.34mm and 2.09mm ( Figure 12 ), and the fresh weight of each plant was 0.92g, 0.90g and 1.52g respectively ( Figure 13), and the dry weight of each plant was 0.29g, 0.29g and 0.40g respectively ( Figure 14 Compared with sterile water treatment, LYZ1125 treatment increased plant height by approximately 30%, compound leaf number by 63%, main stem diameter by 21%, root length by 26%, taproot diameter by 59%, individual plant fresh weight by approximately 64%, and individual plant dry weight by approximately 39%. This indicates that infection with Verticillium alfalfa LYZ0257 significantly impacts alfalfa plants, but the biocontrol agent LYZ1125 can mitigate the pathogen's effects on alfalfa plants.

[0127] from Figure 15 Figure 16 and Figure 17 As shown, after treatment with sterile water (CK), LB culture medium, and 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 efficacy of LB culture medium and B. amyloliquefaciens LYZ1125 against alfalfa Verticillium wilt was 1.3% and 82.9%, respectively. This indicates that 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. It will be apparent to those skilled in the art that the detailed disclosure above is merely illustrative and does not limit 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 and remain within the spirit and scope of the exemplary embodiments of the present application.

[0129] At the same time, this application uses specific terms to describe the embodiments of this 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 this application. Therefore, it should be emphasized and noted that "one embodiment," "an embodiment," or "an alternative embodiment" mentioned twice or multiple times in different locations 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 may be appropriately combined.

Claims

1. Bacillus amyloliquefaciens LYZ1125, characterized in that The Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens LYZ1125 was deposited in the China General Microbiology Culture Collection Center (CGMCC) with the deposit number CGMCC No. 31716.

2. A biocontrol agent, characterized in that: The biocontrol agent includes the Bacillus amyloliquefaciens LYZ1125 according to claim 1.

3. The biocontrol agent according to claim 2, wherein: 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.

4. Use of the biocontrol agent according to claim 2 in preventing and controlling alfalfa diseases, wherein the alfalfa disease is alfalfa verticillium wilt; in, The pathogen of alfalfa verticillium wilt is Verticillium truncatula ( Verticillium alfalfae )LYZ0257.

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

Images