A Velez Bacillus strain QZ-Q2-12 and its application

Abstract

The present invention discloses a Velez subtilis strain QZ-Q2-12 and an application thereof. The strain QZ-Q2-12 is classified and named as Velez subtilis ( Bacillus velezensis ), deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on June 7, 2023, with the deposit number CGMCC No. 27575. The Bacillus Velez strain QZ‑Q2‑12 screened by the present invention has a broad-spectrum inhibitory activity against crop diseases, and has good inhibitory activity against pathogenic oomycetes, pathogenic fungi or pathogenic bacteria. Root irrigation with live QZ‑Q2‑12 bacteria, or coating potato seed potatoes with a biological coating agent containing QZ‑Q2‑12, can significantly promote the growth of potatoes; coating potato seed potatoes with a biological coating agent containing QZ‑Q2‑12 has a control effect of 61.5% on potato late blight.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and relates to a Velezacacillus strain QZ-Q2-12 separated from a potato field in Qingzhen City, Guiyang City, Guizhou Province, China, and an application thereof, and the application thereof in promoting crop growth and preventing and controlling crop diseases. Background Art

[0002] Around the world, crop diseases cause severe economic losses due to reduced yields. Even when growing disease-resistant varieties, growers around the world still lose more than 10% of their crops to diseases each year. Chemical pesticides are mostly used in disease prevention and control, but the long-term irrational use of chemical pesticides has brought serious problems. Among them, the "3R" problem of pesticide residues, resurgence of pests, and resistance of organisms has a great impact on the quality and safety of agricultural products.

[0003] Beneficial microorganisms exist in the crop growth environment, some of which can promote plant growth and / or induce plant resistance to resist disease infection. Using beneficial microorganisms for disease prevention and control is an eco-friendly and sustainable crop disease prevention and control measure. At present, more and more beneficial microorganisms and their related products are used in the fields of biofertilizers and biopesticides. Summary of the invention

[0004] The present invention aims to provide a Velez Bacillus strain QZ-Q2-12 and its application. The strain has a broad-spectrum inhibitory activity against crop diseases and can be used as a microbial coating agent to significantly promote potato growth and prevent potato late blight.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] In the first aspect, the present invention claims a strain of Bacillus Velezii QZ-Q2-12, which is classified and named as Bacillus Velezii ( Bacillus velezensis ), deposited in the General Microbiology Center of China Microorganism Culture Collection on June 7, 2023, with the deposit number CGMCC No. 27575, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The Velez Bacillus strain QZ-Q2-12 of the present invention was isolated from potato field soil, and the strain was identified as Velez Bacillus ( Bacillus velezensis ), 16S rDNA gene sequence as SEQ ID NO.1.

[0008] In a second aspect, the present invention seeks to protect the use of the above-mentioned Bacillus Velez strain QZ-Q2-12 in the following (1) or (2):

[0009] (1) Promoting crop growth and / or preventing and controlling crop diseases;

[0010] (2) Preparation of microbial preparations for promoting crop growth and / or preventing and controlling crop diseases.

[0011] Furthermore, the microbial preparation is live bacteria, fermentation products or metabolites of the Bacillus Velez subtilis strain QZ-Q2-12, or a microbial coating agent or microbial agent made from the Bacillus Velez subtilis strain QZ-Q2-12.

[0012] Further, in the above application, the microbial preparation made from the Velez subtilis strain QZ-Q2-12 is applied to the propagation material of the crop or the crop is treated by drip irrigation and / or spraying to promote crop growth or / and prevent and control crop diseases. Further, the propagation material of the crop is the tuber or seed of the crop. Still further, in a specific embodiment of the present invention, the microbial coating agent made from the Velez subtilis strain QZ-Q2-12 is applied to the propagation material such as the tuber and seed of the crop to promote crop growth or / and prevent and control crop diseases; or the live bacteria, fermentation products or metabolites of the Velez subtilis strain QZ-Q2-12, or the microbial agent made from the Velez subtilis strain QZ-Q2-12 is applied to potted plants, greenhouses, fields, etc. by drip irrigation and / or spraying to treat crops, promote crop growth or / and prevent and control crop diseases.

[0013] Further, the crop diseases are caused by at least one of pathogenic oomycetes, pathogenic fungi and pathogenic bacteria; the pathogenic oomycetes are at least one of Phytophthora infestans and Phytophthora sojae; the pathogenic fungi are at least one of Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium oxysporum, Fusarium equisetum and Fusarium solani; the pathogenic bacteria are potato scab pathogens. Studies have shown that the live bacteria of the Bacillus Velezii QZ-Q2-12 strain screened by the present invention have strong inhibitory activity against the above-mentioned multiple pathogens.

[0014] Furthermore, the crop is potato, but is not limited thereto.

[0015] In a third aspect, the present invention seeks to protect a microbial coating agent, wherein the microbial coating agent comprises the above-mentioned Bacillus Velez strain QZ-Q2-12.

[0016] Furthermore, the microbial coating agent comprises the following ingredients, measured by weight percentage: 10%-20% of the fermentation broth of the above-mentioned Bacillus Velez strain QZ-Q2-12, 1%-3% of a film-forming agent, 1% of a thickener, 0.3% of a colorant, 0.5% of a preservative, 5% of an antifreeze agent, and the remainder being water.

[0017] Further, the spore concentration in the fermentation broth of the Velez Bacillus strain QZ-Q2-12 is 10 9 -10 10 CFU / ml; the film-forming agent is polyvinyl alcohol or sodium carboxymethyl cellulose; the thickener is sodium alginate or chitosan; the colorant is acid fuchsin, new carmine or brilliant blue; the preservative is sodium benzoate or potassium sorbate; the antifreeze agent is propylene glycol or glycerol.

[0018] Studies have shown that the use of live bacteria of Bacillus Velezii strain QZ-Q2-12 to irrigate the roots of potatoes after emergence has an average promotion effect of 45.7% on potato plant height. The above-mentioned microbial coating agent was used to coat potato tubers at a microbial coating agent / seed mass of 1:300, which did not affect emergence, and at the same time, the average promotion effect on potato plant height reached 30.4%. The above-mentioned microbial coating agent has a good preventive effect on late blight caused by Phytophthora infestans, and the control effect reached 61.5%.

[0019] Beneficial effects of the present invention:

[0020] The Velez Bacillus strain QZ-Q2-12 screened by the present invention has a broad-spectrum inhibitory activity against crop diseases, and has good inhibitory activity against pathogenic oomycetes Phytophthora infestans and Phytophthora sojae, pathogenic fungi Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium oxysporum, Fusarium equisetum and Fusarium solani, and pathogenic bacteria Potato scab pathogen. Root irrigation with live bacteria QZ-Q2-12, or coating potato seed potatoes with a microbial coating agent containing QZ-Q2-12, can significantly promote the growth of potatoes; coating potato seed potatoes with a microbial coating agent containing QZ-Q2-12 has a control effect of 61.5% on potato late blight. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The antibacterial activity of live bacteria of strain QZ-Q2-12 against pathogenic oomycetes, fungi and bacteria.

[0022] Figure 2 This is the effect of the live bacteria of strain QZ-Q2-12 on potato plant height.

[0023] Figure 3 The effect of the microbial coating agent made from strain QZ-Q2-12 on potato plant height.

[0024] Figure 4 The microbial coating agent made from strain QZ-Q2-12 has a preventive and control effect on potato late blight. DETAILED DESCRIPTION

[0025] In order to make the purpose and advantages of the present invention clearer, the present invention is now described in detail in conjunction with the following examples. The following examples are only used to more clearly illustrate the technical solutions and contents of the present invention, and cannot be used to limit the protection scope of the present invention. Any modifications, equivalent replacements and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

[0026] Embodiment 1:

[0027] The researchers of the present invention isolated and screened a strain QZ-Q2-12 with broad-spectrum inhibitory activity against crop diseases from potato field soil in Qingzhen City, Guiyang City, Guizhou Province, China, and performed PCR amplification, sequence determination and comparison on the 16S rRNA gene of the strain QZ-Q2-12.

[0028] The composition of LB medium is: 10 g Tryptone, 10 g NaCl, 5 g Yeast extract, pH 7.0, add 1000 ml sterile water, mix well, and sterilize in aliquots.

[0029] The strain was inoculated into liquid LB medium, shaken at 200 rpm and 37℃ for 12 h, and the bacteria were collected by centrifugation. The genomic DNA was extracted using the bacterial extraction kit of Tiangen Company. The 16S rDNA gene was amplified by PCR using the forward primer 27F (5'-AGA GTT TGA TCC TGG CTCAG-3') and the reverse primer 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3'). The amplification of the 16S rRNA fragment was observed by gel electrophoresis and the PCR stock solution was sent for sequencing. The PCR conditions were: 95℃, 4min; 94℃, 1 min; 54℃, 1 min; 72℃, 1 min; 32 cycles; 72℃, 10 min, and stored at 4℃. The length of the 16S rDNA partial gene sequence was about 1500 bp, and the result is shown in SEQ ID NO.1.

[0030] SEQ ID NO.1 was compared on the NCBI database website, and the comparison results are shown in Table 1. According to the results in Table 1, it can be inferred that the strain QZ-Q2-12 of the present invention is Bacillus velezinis ( Bacillus velezensis ).

[0031] Table 1 Sequence alignment results of SEQ ID NO.1 in the public website NCBI

[0032] .

[0033] Example 2: Bacillus Velez Bacillus velezensis ) The live bacteria of strain QZ-Q2-12 have inhibitory activity against pathogenic oomycetes, fungi and bacteria

[0034] Bacillus Velez Bacillus velezensis ) The common fermentation culture method of strain QZ-Q2-12 is: inoculate strain QZ-Q2-12 into LB liquid culture medium for large-scale shaking culture at a temperature of 37°C, a shaking speed of 200 rpm, and a fermentation time of 12 hours.

[0035] The plate confrontation method was used to determine the inhibitory activity of live bacteria of strain QZ-Q2-12 against pathogenic oomycetes, fungi and bacteria. The selected pathogenic oomycetes were Phytophthora infestans and Phytophthora sojae; the pathogenic fungi were at least one of Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium oxysporum, Fusarium equisetum and Fusarium solani; the pathogenic bacteria were potato scab pathogens. The determination method was the plate confrontation method. The experimental method for oomycetes and fungi was to place a pathogenic bacteria disc with a diameter of 1 cm in the center of the PDA plate, and measure the OD value at a distance of 2 cm on both sides of the plate. 600 =1; the bacterial assay method is to mix the bacterial solution with the culture medium at a volume ratio of 1:100, prepare the NA plate containing pathogens, and drop 10 μl of the Bacillus solution (OD 600 =1). The test results showed that the live bacteria of strain QZ-Q2-12 had good antibacterial effects on Phytophthora infestans, Phytophthora sojae, Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium oxysporum, Fusarium equisetum, Fusarium solani and potato scab pathogen. Figure 1 shown.

[0036] Example 3: Bacillus Velez Bacillus velezensis ) The live bacteria of strain QZ-Q2-12 have a promoting effect on potato plant height

[0037] Potato variety Holland 15 was selected, cut into pieces of about 50 g and sown into soil containing nutrient soil and vermiculite (mixed in a mass ratio of 1:1) in pots with a diameter of 10 cm. One potato tuber was sown in each pot, and three pots were made for each treatment. The strain QZ-Q2-12 was inoculated into LB liquid culture medium for large-scale shaking culture at a temperature of 37°C and a shaking speed of 200 rpm. The fermentation time was 12 hours, and the samples were taken out for measurement and diluted to OD 600 =1. After the potatoes emerged, pots with uniform growth were selected and roots were irrigated with 50 ml of fermentation solution. LB medium was used as a control. Each treatment was repeated three times. After 38 days of growth, the length of potato stems was measured. Figure 2As shown in Table 2, the plant height of the QZ-Q2-12 live bacteria root irrigation group increased by 45.7% compared with the control group.

[0038] Table 2 Statistical results of plant height and growth rate

[0039] .

[0040] Example 4: Bacillus Velez Bacillus velezensis ) The microbial agent made from strain QZ-Q2-12 has a promoting effect on potato plant height

[0041] The strain QZ-Q2-12 was prepared into a microbial coating agent according to the following steps.

[0042] Preparation Example 1: The fermentation broth of Bacillus Velezii strain QZ-Q2-12 (spore concentration 10 9 -10 10 CFU / ml) 15 wt %, film-forming agent polyvinyl alcohol 2 wt %, thickener sodium alginate 1 wt %, colorant acid fuchsin 0.3 wt %, preservative sodium benzoate 0.5 wt %, antifreeze agent propylene glycol 5 wt %, the rest is water to make up 100%, and the mixture is stirred and mixed evenly.

[0043] Preparation Example 2: The fermentation broth of Bacillus Velezii QZ-Q2-12 strain (spore concentration 10 9 -10 10 CFU / ml) 10 wt %, film-forming agent sodium carboxymethyl cellulose 1 wt %, thickener sodium alginate 1 wt %, colorant acid fuchsin 0.3 wt %, preservative sodium benzoate 0.5 wt %, antifreeze agent propylene glycol 5 wt %, the rest is water to make up 100%, and the mixture is stirred and mixed evenly.

[0044] Preparation Example 3: The fermentation broth of Bacillus Velezii QZ-Q2-12 strain (spore concentration 10 9 -10 10 CFU / ml) 20 wt %, film-forming agent sodium carboxymethyl cellulose 3 wt %, thickener chitosan 1 wt %, colorant acid fuchsin 0.3 wt %, preservative sodium benzoate 0.5 wt %, antifreeze agent propylene glycol 5 wt %, the rest is water to make up 100%, and the mixture is stirred and mixed evenly.

[0045] Select potato variety Holland 15 and cut into pieces of about 50 g. Coat the seed potatoes with the microbial agent prepared in Preparation Example 1 at a mass ratio of 300:1, and sow after drying. Select a pot with a diameter of 10 cm and sow in soil filled with nutrient soil and vermiculite (mixed in a mass ratio of 1:1). Sow one potato tuber in each pot, with three replicates for each treatment, and use the uncoated tuber as a control. Measure the length of the potato stem after 39 days of growth. Figure 3 As shown in Table 3, the QZ-Q2-12 coating agent treatment had no effect on seedling emergence compared with the control group, and the plant height increased by 30.4%.

[0046] Table 3 Statistical results of germination rate, plant height and growth rate

[0047] .

[0048] Example 5: Bacillus Velez Bacillus velezensis ) Effect of microbial agents made from strain QZ-Q2-12 on the prevention and control of potato late blight

[0049] Select the potato variety Holland 15 and cut it into pieces of about 50 g. The seed potatoes and the microbial agent prepared in Preparation Example 1 of Example 4 are coated at a mass ratio of 300:1, and sowed after drying. Select a pot with a diameter of 10 cm and sow in soil filled with nutrient soil and vermiculite (mixed in a mass ratio of 1:1). Sow one potato tuber in each pot, with three replicates for each treatment, and use the uncoated tuber as a control. After germination, grow for another month, spray about 5 ml of zoospores (concentration 50 / ul), and then put it in a transparent plastic bag to keep it moist for three days. Remove the plastic bag after three days, and calculate the disease index after another four days. Figure 4 As shown in the figure, QZ-Q2-12 bacterial agent coating has better prevention effect on potato late blight. The protection effect of the bacterial agent after coating is 61.5%, which is significantly higher than the coating treatment of the published patented strain DMW1 (CGMCC No. 23191, ZL 202111311436.9) (28.5%).

[0050] Table 4 Statistical results of emergence rate, disease index and control effect

[0051] .

[0052] Sequence Listing

[0053] SEQ ID No.1

[0054]

Claims

1. A strain of Bacillus Velezii QZ-Q2-12, which is classified and named as Bacillus Velezii ( Bacillus velezensis ), deposited in the General Microbiology Center of China Microorganism Culture Collection on June 7, 2023, with the deposit number CGMCC No. 27575, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The use of the Bacillus Velez strain QZ-Q2-12 according to claim 1 in promoting crop growth or / and preventing and controlling crop diseases, characterized in that: The crop disease is a crop disease caused by at least one of pathogenic oomycetes, pathogenic fungi and pathogenic bacteria; the pathogenic oomycetes is at least one of Phytophthora infestans and Phytophthora sojae; the pathogenic fungi is at least one of Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium oxysporum, Fusarium equisetum and Fusarium solani; the pathogenic bacteria is potato scab pathogen; and the crop is potato.

3. Use of the Bacillus Velez strain QZ-Q2-12 according to claim 1 in the preparation of a microbial preparation for promoting crop growth or / and preventing and controlling crop diseases, characterized in that: The crop disease is a crop disease caused by at least one of pathogenic oomycetes, pathogenic fungi and pathogenic bacteria; the pathogenic oomycetes is at least one of Phytophthora infestans and Phytophthora sojae; the pathogenic fungi is at least one of Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium oxysporum, Fusarium equisetum and Fusarium solani; the pathogenic bacteria is potato scab pathogen; and the crop is potato.

4. The use according to claim 3, characterized in that: The microbial preparation is the live bacteria of the Velez subtilis strain QZ-Q2-12, or a microbial coating agent or a microbial agent made from the Velez subtilis strain QZ-Q2-12.

5. The use according to claim 2, characterized in that: The microbial preparation prepared by the Velez Bacillus strain QZ-Q2-12 is applied to crop propagation materials or crops are treated by drip irrigation and / or spraying to promote crop growth and / or prevent and control crop diseases.

6. A microbial coating agent, characterized in that: The microbial coating agent comprises the Bacillus Velez subtilis strain QZ-Q2-12 described in claim 1.

7. The microbial coating agent according to claim 6, characterized in that The microbial coating agent comprises the following ingredients by weight percentage: 10%-20% of the fermentation broth of the Bacillus Velez subtilis strain QZ-Q2-12 according to claim 1, 1%-3% of a film-forming agent, 1% of a thickener, 0.3% of a colorant, 0.5% of a preservative, 5% of an antifreeze agent, and the remainder is water.

8. The microbial coating agent according to claim 7, characterized in that The spore concentration in the fermentation broth of the Velez Bacillus strain QZ-Q2-12 is 10 9 -10 10 CFU / ml; The film-forming agent is polyvinyl alcohol or sodium carboxymethyl cellulose; The thickener is sodium alginate or chitosan; The colorant is acid fuchsin, new carmine or brilliant blue; The preservative is sodium benzoate or potassium sorbate; The antifreeze agent is propylene glycol or glycerol.

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