Bacillus Velezii QN2320, biocontrol agent and application thereof

Abstract

The invention discloses a strain of Bacillus Velez QN2320, a biocontrol agent and an application thereof. The Bacillus Velez QN2320 is deposited in the China Center for Type Culture Collection, and its deposit number is CCTCC NO: M 20241601. The invention also discloses a biocontrol agent, whose active ingredients include the bacterial body, bacterial liquid, fermentation filtrate or active extract of the Bacillus Velez QN2320. The Bacillus Velez has excellent antagonistic effects on various plant disease pathogens such as Diplodia pine, Fusarium graminearum and Cordyceps sinensis, has a broad-spectrum antibacterial property, and has high antibacterial activity. It can be used as a biocontrol agent for the prevention and treatment of various plant diseases such as pine trunk rot, wheat stem base rot, apple rot, and apple ring rot, and has good market application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus velez QN2320, a biocontrol agent and applications thereof. Background Art

[0002] Sustained agricultural production and harvest is a decisive factor in my country's food security. However, crops are easily infected by pathogens during their growth and may even suffer from diseases under suitable conditions, causing huge losses to agricultural production. With global warming, the outbreak and prevalence of crop diseases and insect pests will become more frequent. It is predicted that by the end of this century, the incidence of crop diseases and insect pests will increase by 243-460%. Diseases have become an important limiting factor for stable and high yields of crops.

[0003] In order to effectively prevent and control plant diseases and reduce losses to agricultural production and forestry, many plant disease and insect pest control technology systems have been established. However, in the practice of disease and insect pest control, the application of chemical pesticides still dominates the market, accounting for 80%. The excessive use of chemical pesticides has caused many negative effects, such as ecological damage, environmental pollution, excessive residues, resistance of pathogens, and even threatens human health. In order to promote the green development of agricultural production and reduce the use of chemical pesticides, it is urgent to develop and promote green plant disease and insect pest control technologies. Our country has begun to develop and promote the application of technologies to reduce the use of chemical pesticides and chemical fertilizers in agricultural production.

[0004] Biological control is an important measure for plant disease prevention and control. Biocontrol bacteria can effectively prevent and control plant diseases and insect pests by inducing plant resistance, competition for nutrition and ecological niches, antagonism, parasitism, and bacteriolysis. In addition, biocontrol agents are environmentally friendly, ecologically safe, and harmless to natural enemies. Biocontrol agents prepared using antagonistic bacteria are the best alternative to chemical agents. It can be seen that biological control measures are the development direction and important means of plant disease prevention and control. They have very broad application prospects in agricultural production and are of great significance to ensuring the healthy and sustainable development of agricultural production.

[0005] At present, there are many commercialized biocontrol agents in the world, which have been widely used in the prevention and control of plant diseases such as plant virus diseases, plant anthracnose, and plant nematode diseases. The patent with the publication number CN112980721B previously applied by the applicant disclosed a strain of Bacillus Velez QBB3. The conidia of Pseudomonas graminearum can still germinate in the fermentation filtrate of 10% QBB3. Therefore, Bacillus Velez QBB3 needs a higher concentration when applied, and the cost is high. Biocontrol bacteria with higher antibacterial activity need to be developed.

[0006] Therefore, the existing technology needs to be further improved. Summary of the invention

[0007] In view of the above problems, the present invention provides a strain of Bacillus Velezii QN2320, a biocontrol agent and applications thereof. The strain has a broad antibacterial spectrum and strong antibacterial activity, and can be widely used in the prevention and treatment of various plant disease pathogens.

[0008] To solve the above problems, the technical solution of the present invention is as follows:

[0009] In the first aspect, the present application provides a strain of Bacillus velezensis, which is named Bacillus velezensis QN2320. The bacterium was deposited in the China Center for Type Culture Collection on July 18, 2024, with a deposit number of CCTCC NO: M 20241601, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0010] The Bacillus velezensis QN2320 was isolated from the soil of Jiaozhou Science and Technology Demonstration Park in Qingdao. The molecular biological identification results showed that the strain QN2320 and Bacillus velezensis CBMB205 were clustered together, indicating that QN2320 is a strain of Bacillus velezensis. Combining the morphological characteristics with the molecular biological identification results, the strain was determined to be Bacillus velezensis and named QN2320.

[0011] In a second aspect, the present application also provides a biocontrol agent, the active ingredient of which includes the aforementioned Bacillus Velez QN2320.

[0012] Optionally, in the biocontrol agent, the Bacillus Velez subtilis is the bacterial body, bacterial liquid, fermentation filtrate or active extract of Bacillus Velez subtilis QN2320.

[0013] Optionally, the biocontrol agent further comprises other biocontrol bacteria used in combination with Bacillus Velez QN2320. In order to further optimize the bactericidal effect of the biocontrol agent and further expand the antibacterial range of the biocontrol agent, other biocontrol bacteria that do not have antagonism can be used in combination with Bacillus Velez QN2320 to give play to the complementary advantages of multiple groups of biocontrol bacteria and improve the control effect of the biocontrol bacteria.

[0014] The liquid or solid biocontrol agent can be prepared by mixing the bacterial body, bacterial liquid, fermentation filtrate or active extract of Bacillus Velez QN2320 with other auxiliary agents and adsorbents.

[0015] In a third aspect, the present application also provides the use of the aforementioned Bacillus Velezii or the aforementioned biocontrol agent in preventing and controlling plant diseases.

[0016] Optionally, in the application, the plant diseases include: pine trunk rot, wheat stem base rot, apple rot, apple ring rot, grape gray mold, grape anthracnose and tomato early blight.

[0017] Optionally, the plant disease pathogens include: Diplodia sapinea (D. sapinea), Fusarium pseudograminearum (F. pseudograminearum), V. mali (V. mali), B. dothidea, B. cinerea, C. gloeosporioides, and A. solani.

[0018] The experimental results show that Bacillus Velez QN2320 has excellent antagonistic effects on the above-mentioned various plant disease pathogens, among which the antibacterial effect on Diplodia pine, Fusarium graminearum and Cordyceps sinensis is the highest. The antibacterial rate of 10% Bacillus Velez QN2320 filtrate against Diplodia pine, Fusarium graminearum and Cordyceps sinensis is as high as 100%, which is significantly better than the existing biocontrol bacteria Bacillus Velez QBB3.

[0019] Optionally, in the above application, the application to plant diseases includes: inhibition of conidia germination and inhibition of hyphae growth. Experiments have shown that the control effect of Bacillus Velezii QN2320 is reflected in the inhibition of conidia germination and hyphae growth of pathogens, thereby exerting an excellent antibacterial effect. In the embodiments of the present application, Pseudomonas graminearum is used as an example for illustration, but it is not limited to this pathogen.

[0020] In a fourth aspect, the present application also provides a method for preventing and controlling plant diseases, which comprises applying the bacterial solution of the aforementioned Bacillus Velez subtilis or the aforementioned biocontrol agent to the diseased plants.

[0021] The present invention has the following beneficial effects:

[0022] 1. The present invention provides a novel broad-spectrum bacillus Velez QN2320, which has excellent antagonistic effects on pathogens of various common plant diseases such as pine trunk rot, wheat stem base rot, apple rot, apple ring rot, grape gray mold, grape anthracnose and tomato early blight. Among them, the bacteriostasis of the bacterium against D. sapinea (the pathogen of pine trunk rot), F. pseudograminearum (the pathogen of wheat stem base rot) and V. mali (the pathogen of apple rot) is the highest. The bacteriostasis rate of 10% of the filtrate of Bacillus Velez QN2320 against these three pathogens is as high as 100%, which is significantly better than the existing biocontrol bacterium Bacillus Velez QBB3. It can be prepared into a biocontrol agent, which has a good market application prospect.

[0023] 2. The inhibitory effect of Bacillus Velez QN2320 is diversified, which is not only reflected in the inhibition of the growth of pathogenic hyphae, but also in the inhibition of the normal germination of pathogenic conidia, thus exerting an excellent antibacterial effect as a whole.

[0024] 3. The QBB1 strain and its biocontrol agent provided by the present invention provide a new method for the prevention and treatment of important plant pathogenic bacteria diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Phylogenetic relationship analysis of 16S rDNA of Bacillus velez QN2320;

[0026] Figure 2 The inhibition of QN2320 fermentation filtrate on the colony growth of Diplodia pinecolor; wherein A is the colony formed by Diplodia pinecolor on PDA; B: Diplodia pinecolor cannot grow on PDA containing 5% QN2320 fermentation filtrate;

[0027] Figure 3 is the inhibition of the growth of the colony of Pseudofusorium graminearum by the fermentation filtrate of QN2320; wherein A is the colony formed by Pseudofusorium graminearum on PDA, B is the colony formed by Pseudofusorium graminearum on PDA with 5% of the fermentation filtrate of QN2320, and C is the colony formed by Pseudofusorium graminearum on PDA with 10% of the fermentation filtrate of QN2320;

[0028] Figure 4 The inhibition of conidia germination of F. graminearum by QN2320 fermentation filtrate; A: normal germination of conidia of F. graminearum; B: conidia of F. graminearum treated with 2% QN2320 fermentation filtrate;

[0029] Figure 5The control effect of QN2320 fermentation filtrate on wheat stem base rot; A is wheat seedlings inoculated with conidia of Pseudomonas graminearum, B is wheat seedlings inoculated with conidia of Pseudomonas graminearum and then treated with QN2320 fermentation filtrate, and C is wheat seedlings in the blank control group treated with LB. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0031] Example 1 Isolation and Identification of Bacillus Velezii QN2320

[0032] 1. Isolation and screening of Bacillus velez QN2320

[0033] (1) 56 strains of Bacillus were isolated from the soil in Jiaozhou Science and Technology Demonstration Park, Qingdao in May 2022. The isolation method is as follows: collect field soil samples, take 10g of soil and add it to 90ml of sterile water, vortex and let it stand for 30 minutes, take the supernatant and sterilize it with wet heat at 121℃ for 10 minutes, then streak it on solid LB medium, and pick a single colony after 2 days.

[0034] (2) Through plate confrontation culture, a strain was screened out that has strong inhibitory effects on multiple pathogens, including D. sapinea (the pathogen of pine trunk rot), F. pseudograminearum (the pathogen of wheat stem rot), and V. mali (the pathogen of apple rot).

[0035] 2. Morphological identification

[0036] The screened strains formed round milky white colonies on solid LB medium, and the edges of the colonies were not very neat and easily wrinkled.

[0037] 3. Molecular identification of QN2320

[0038] (1) Identification method:

[0039] A single colony of QN2320 was inoculated into liquid LB medium for cultivation, and then centrifuged and the bacteria were collected to extract the genomic DNA of the bacteria. The 16S rDNA of QN2320 was amplified using 27F / 1492R as primers, and the obtained DNA fragments were sequenced. The 16S rDNA sequence of the bacteria obtained by sequencing was subjected to phylogenetic analysis using MEGA (X). The primer sequences are as follows:

[0040] 27F:TACGGYTACCTTGTTACGACTT

[0041] 1492R: AGAGTTTGATCMTGGCTCAG.

[0042] (2) Results and analysis

[0043] The sequencing results of 16S rDNA of QN2320 are shown in SEQ ID NO: 1 in the sequence table. The results of phylogenetic analysis are shown in Figure 1 It can be seen that QN2320 and Bacillus velezensis CBMB205 are clustered into one branch, and the sequence homology between the two is 99.58%, indicating that QN2320 belongs to Bacillus velezensis.

[0044] Therefore, the results of morphological and molecular identification showed that the strain was a new strain of Bacillus velezinsis, which was named QN2320.

[0045] Example 2 Inhibition of QN2320 on various plant disease pathogens

[0046] 1. Experimental methods

[0047] (1) Preparation of QN2320 fermentation filtrate

[0048] A single colony of QN2320 was streaked on LB solid medium and shaken in small quantities. According to the inoculation amount of 2%, 100 mL of LB liquid medium was loaded into a 250 mL Erlenmeyer flask, shaken at 28°C and 160 rpm for 72 hours, and filtered through a bacterial filter (diameter 0.22 um) to obtain the fermentation filtrate of QN2320. The concentration of the fermentation filtrate was diluted to 5% and 10% respectively for subsequent experiments.

[0049] (2) Antagonism of QN2320 against plant disease pathogens

[0050] The antibacterial rates of 5% and 10% QN2320 filtrate against various plant pathogens were tested by the plate confrontation culture method.

[0051] The specific experimental method is: the fermentation filtrate of QN2320 is added to the PDA medium to a final concentration of 5% and 10%, respectively, and the surface of the PDA medium is inoculated with a newly activated pathogen cake.

[0052] The calculation method of the antibacterial rate is:

[0053] Inhibition rate = (control colony radius - treated colony radius) / control colony radius x 100%

[0054] 2. Experimental results and analysis

[0055] The experimental results are shown in Table 1 below. It can be seen from the table that QN2320 has excellent antibacterial effects on the pathogens of the following seven plant diseases: D. sapinea, F. pseudograminearum, V. mali, B. dothidea, B. cinerea, C. gloeosporioides, and A. solani. Among them, the antibacterial effect on D. sapinea, F. pseudograminearum, and A. solani is the highest. The antibacterial rate of 10% QN2320 filtrate on D. sapinea, F. pseudograminearum, and A. mali is as high as 100%, which is significantly better than the effect of the existing biocontrol bacterium Bacillus velez.

[0056] Table 1 Antagonistic effect of QN2320 on plant disease pathogens

[0057] Plant diseases Pathogens 5% filtrate antibacterial rate (%) 10% filtrate antibacterial rate (%) Pine trunk rot D.sapinea 100 100 Wheat stem rot F. pseudograminearum 76.00 100 Apple rot V.mali 97.06 100 Apple ring rot B.dothidea 85.30 95.60 Botrytis cinerea B. cinerea 73.53 82.35 Grape Anthracnose C. gloeosporioides 61.76 76.47 Tomato Early Blight A.solani 76.47 82.35

[0058] Example 3 Analysis of the specific inhibition of pathogens by QN2320

[0059] 1. Inhibitory effect of QN2320 fermentation filtrate on the growth of D. sapinea

[0060] (1) Experimental methods:

[0061] The bacterial cakes (0.5 cm in diameter) of Diplodia pine were inoculated into PDA medium with 0, 1%, 2%, 5% and 10% of the fermentation filtrate of QN2320, respectively, and cultured at 25° C. When the colonies of Diplodia pine on the PDA medium with 0% of the fermentation filtrate filled the dish, the results were tested.

[0062] (2) Experimental results

[0063] The results are as follows Figure 2 As shown, the results indicate that 5% of the fermentation filtrate completely inhibited the growth of the Pinus sphaeroides colonies.

[0064] 2. Inhibitory effect of QN2320 fermentation filtrate on the growth of F. pseudograminearum

[0065] (1) Experimental methods

[0066] According to the method for determining the growth inhibition of QN2320 fermentation filtrate on Diplodia pine, the inhibitory effect of QN2320 fermentation filtrate on the growth of Fusarium graminearum was determined.

[0067] (2) Experimental results and analysis

[0068] The results are as follows Figure 3 The results show that on PDA containing 10% of QN2320 fermentation filtrate, the hyphae of Pseudofusorium graminearum cannot grow on the PDA medium and can only form aerial hyphae, indicating that 10% fermentation filtrate completely inhibits the growth of Pseudofusorium graminearum colonies.

[0069] 3. Inhibitory effect of QN2320 fermentation filtrate on conidia germination of Fusarium graminearum

[0070] (1) Experimental methods:

[0071] The fermentation filtrate of QN2320 was diluted with YEPD liquid medium to a final content of 1%, 2%, 5%, and 10%, and the YEPD liquid medium without addition was used as the control. The control and each test group were treated with conidia of Pseudofus graminearum, cultured at 25°C, and the results were detected when the conidia germination rate of the control group reached 90%.

[0072] (2) Experimental results and analysis

[0073] The experimental results show that when the concentration of the fermentation filtrate is 5%, the germination of the conidia of F. graminearum is completely inhibited; when the concentration of the fermentation filtrate is 2%, 6% of the conidia germinate, but the germ tubes cannot develop normally, forming an enlarged spherical structure (such as Figure 4 B).

[0074] Example 4. Control effect of QN2320 fermentation filtrate on wheat stem rot

[0075] 1. Experimental methods

[0076] Pot experiment was used to test the control effect of QN2320 fermentation filtrate on wheat stem rot. The wheat seedlings used were Jimai 22.

[0077] Treatment and control groups were set up with a conidia solution of Pseudomonas graminearum (concentration of 10 5The wheat seedlings in the treatment group were then watered with 20 mL of QN2320 fermentation filtrate, and the control group was watered with the same volume of liquid LB medium.

[0078] The blank control group was not inoculated with F. pseudograminearum, nor treated with QN2320 fermentation filtrate, but was treated with the same amount of LB.

[0079] The wheat seedlings were cultured under the conditions of 95% relative humidity and 25°C, and the disease status of the wheat seedlings was investigated after 2 weeks. The wheat seedling disease index was calculated according to the following formula:

[0080]

[0081] The control effect (%) = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100%.

[0082] 2. Experimental results and analysis

[0083] The results showed that the disease index of the control group inoculated with only pathogen conidia was 78, the disease index of the group treated with QN2320 fermentation filtrate was 7.5, and the disease index of the blank control group was 0. It was calculated that the control effect of QN2320 fermentation filtrate on wheat stem base rot was 90.4%. Figure 5 .

[0084] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A strain of Bacillus velez Bacillus velezensis ) QN2320, characterized in that, Its accession number is CCTCC NO: M 20241601.

2. A biocontrol agent, characterized in that: The active ingredient comprises the Bacillus Velez QN2320 described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that The Bacillus Velez is the bacterial body, bacterial liquid or fermentation filtrate of Bacillus Velez QN2320.

4. The biocontrol agent according to claim 3, characterized in that The biocontrol agent also includes other biocontrol bacteria used in combination with Bacillus Velez QN2320.

5. Use of the Bacillus Velezii according to claim 1 or the biocontrol agent according to claim 2 in preventing and controlling plant diseases; the plant diseases are: pine trunk rot, wheat stem base rot, apple rot, apple ring rot, grape gray mold, grape anthracnose and tomato early blight; the plant disease pathogen is: Diplodia pine Diplodia sapinea ), Pseudomonas graminearum ( Fusarium pseudograminearum )、Black rot fungus( Valsa mali )、Botrytis cinerea( Botryosphaeria dothidea )、Botrytis cinerea( Botrytis cinerea , Colletotrichum gloeosporioides Colletotrichum gloeosporioides ) and Alternaria solani ( Altanaria solani ).

6. The use according to claim 5, characterized in that: Applications to plant diseases include: inhibition of conidia germination and inhibition of mycelium growth.

7. A method for preventing and controlling plant diseases, characterized in that: The control method comprises: applying the bacterial solution of Bacillus Velez QN2320 according to claim 1 or the biocontrol agent according to any one of claims 2 to 4 to the diseased plants; the plant diseases are: pine trunk rot, wheat stem base rot, apple rot, apple ring rot, grape gray mold, grape anthracnose and tomato early blight; the plant disease pathogen is: Diplodia pine Diplodia sapinea ), Pseudomonas graminearum ( Fusarium pseudograminearum )、Black rot fungus( Valsa mali )、Botrytis cinerea( Botryosphaeria dothidea )、Botrytis cinerea( Botrytis cinerea , Colletotrichum gloeosporioides Colletotrichum gloeosporioides ) and Alternaria solani ( Altanaria solani ).

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