A strain of Bacillus Velezii and its application in controlling garlic leaf blight

Abstract

The invention discloses a strain of Bacillus velezensis, which is named JSBV63, isolated from nature and deposited with CGMCC No.24638; the microbial agent prepared by the Bacillus velezensis belongs to a biological agent, avoids the problems caused by the use of chemical control agents, and is beneficial to the pollution-free green production of garlic. The JSBV63 strain can significantly inhibit the growth of Stipes stolonifer, has a good inhibitory effect on it, and has a good control effect on garlic leaf blight caused by Stipes stolonifer, can effectively control the occurrence of garlic leaf blight, has a good disease prevention effect, and has a great potential to be developed into a commercial live bacterial biocontrol agent.

Description

Technical Field

[0001] The invention relates to the technical field of crop biocontrol strains, in particular to a Velezius sp. JSBV63 and application thereof in preventing and controlling garlic leaf blight. Background Art

[0002] Garlic (Allium sativum L) is a vegetable crop belonging to the genus Allium of the Liliaceae family. It is a traditional condiment that people love because of its rich nutrition. China is the main producer of garlic, accounting for 80% of the world's output. However, in recent years, with the development of garlic planting area and industrialization and the continuous changes in the cultivation ecological environment, garlic leaf blight (the pathogen of the disease is Stemphylium solani) has become increasingly serious, causing serious losses in the yield of garlic stalks and garlic heads. Garlic leaf blight occurs to varying degrees in all production areas. The losses caused by garlic leaf blight can reach 20% to 30%, and in severe years and some plots, the losses can even be as high as 60% or more.

[0003] There are many types of Stemphylium, which can also cause plant diseases such as tomato gray leaf spot. Tomato Stemphylium is basically Stemphylium lycopersici (Enjoji) Yamamoto, which can be cultured on PDA culture medium. The fungus grows quickly, produces transparent radial hyphae in 1-2 days, and begins to produce yellow pigment in 3-4 days. It can spread on the culture medium and generally grows all over the culture medium plate in 7-10 days, but cannot produce spores on common artificial culture media. So far, there is no clear report of spore production method; the pathogen of tomato gray leaf spot mainly destroys tomato leaves: when the leaves are infected by the disease, gray-brown nearly circular spots with a long diameter of 2-4mm appear on the surface of the leaves, and the spots gradually expand along the veins to form irregular shapes, and dry up and are easy to perforate in the later stage. However, the Stemphylium solani that causes garlic leaf blight can be cultured on PDA culture medium but does not produce yellow pigment. Garlic leaf blight mainly harms leaves: the disease mostly starts from the tips of the lower leaves. In the early stage of the disease, the lesions are water-soaked, the leaf color gradually fades, and grayish-white slightly concave circular spots appear on the leaf surface. As the lesions expand, they turn grayish-yellow to grayish-brown, and purple-black when the air humidity is high. This shows that the Stemphylium pathogens of different plants are different, and the diseases they cause are also different.

[0004] At present, the prevention and control of garlic leaf blight mainly relies on chemical control. Although chemical agents can prevent and control garlic leaf blight to a certain extent, the long-term use of a certain drug alone will cause the pathogen to develop drug resistance, which seriously hinders the green and sustainable development of the garlic industry. As an effective means of plant disease prevention and control, biological control is receiving more and more attention. The application of microbial antagonists to plant diseases has shown good disease prevention and control effects on a variety of crops such as cucumbers, peppers, rice, wheat, and corn. However, there are still few studies on the biological control of garlic leaf blight. Therefore, screening antagonistic strains of garlic leaf blight pathogens, especially biological control of garlic leaf blight caused by Stemphylium solani, can improve the level of garlic leaf blight prevention and control and provide solid technical support for the production of green products. Summary of the invention

[0005] In order to overcome at least one problem in the prior art, the present invention separates and screens a strain of Bacillus velezensis JSBV63, which has an excellent control effect on garlic leaf blight.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first object of the present invention is to provide a strain of Bacillus velezensis JSBV63, whose preservation number is CGMCC No. 24638, whose classification name is Bacillus velezensis, and whose preservation date is April 6, 2022. The preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration (referred to as CGMCC), and the preservation unit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, the sequence of 16S rDNA of the Bacillus velezensis JSBV63 is shown in SEQ ID No.1.

[0009] Furthermore, the morphological characteristics of the Velezella JSBV63 are: on the LB solid culture medium, it appears as light yellow opaque colonies with a smooth surface, wrinkled edges, and a slight bulge in the middle.

[0010] The second aspect of the present invention is to provide a microbial agent comprising at least one of the bacterial cells, spores and fermentation broth of Bacillus Velezii described in any one of the first aspect of the present invention.

[0011] Furthermore, the live bacteria concentration of the microbial agent is 5×10 7 ~5×10 8 CFU / mL; preferably 8×10 7 ~2×108 CFU / mL, more preferably 1×10 8 CFU / mL.

[0012] Furthermore, the microbial agent also includes pesticide-acceptable adjuvants, including but not limited to: dispersants, stabilizers, carriers, etc., to make a favorable dosage form or improve stability, activity, etc., and its components can be formulated according to actual needs. The dosage form of the above-mentioned microbial agent includes but is not limited to: liquid preparations, solid preparations (such as powders), etc. It is understandable that the above-mentioned microbial agent can also be used in combination with other biocontrol agents, chemical agents, etc.

[0013] Furthermore, the microbial agent is a fermentation broth obtained by culturing Bacillus Velez subtilis, and the preparation of the fermentation broth includes the steps of: inoculating Bacillus Velez subtilis JSBV63 in LB culture medium, culturing it in the dark for a predetermined time, inoculating it in LB liquid culture medium for fermentation, and culturing it for a predetermined time to obtain the fermentation broth of Bacillus Velez subtilis JSBV63.

[0014] Furthermore, the LB liquid culture medium consists of 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride.

[0015] Furthermore, the dark culture conditions are: 30-37°C, culture for 12-36 hours; preferably, 37°C dark culture for 24 hours.

[0016] Furthermore, the fermentation conditions are: fermentation temperature 30-37°C, fermentation time 48-72h, shaking culture at a rotation speed of 180-220rpm; preferably, shaking culture at a rotation speed of 180rpm / min and a temperature of 37°C in the dark for 48h.

[0017] Furthermore, in a specific embodiment, the fermentation broth preparation step includes: inoculating the strain JSBV63 into LB culture medium for growth, culturing in a 37°C constant temperature incubator in the dark, and setting aside after culturing for 24 hours; inoculating the spare JSBV63 into 1L LB liquid culture medium (the culture medium consists of 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, and 15g of agar, and is made up to 1000ml with distilled water, and sterilized at 121°C for 20min.) for fermentation, and after shaking culturing at a speed of 180rpm / min and a temperature of 37°C in the dark for 48h, the fermentation broth of JSBV63 is obtained, and stored at 4°C for later use.

[0018] The third aspect of the present invention is to provide an application of the Bacillus Velezii described in any one of the first aspect of the present invention, or the microbial agent described in any one of the second aspect of the present invention, which includes at least one of the following applications: application in inhibiting the growth of pathogens, application in preventing and controlling plant diseases caused by pathogens; wherein the pathogen is a pathogen of the genus Stoloniferum, and the plant is a plant of the family Liliaceae.

[0019] Furthermore, the plant disease is garlic leaf blight.

[0020] The fourth aspect of the present invention is to provide a method for inhibiting the growth of pathogenic bacteria of the genus Stolonemosis or a method for preventing and controlling plant diseases caused by pathogenic bacteria of the genus Stolonemosis, which comprises the steps of applying the Bacillus Velezii described in any one of the first aspect of the present invention or the microbial agent described in any one of the second aspect of the present invention to the pathogenic bacteria, the above-ground parts of the plant body or the growth environment of the plant root system.

[0021] Furthermore, the plant disease is garlic leaf blight.

[0022] Furthermore, when potted garlic leaf blight is controlled, the application dosage of the microbial agent is: JSBV63 fermentation liquid (bacteria content is 10 8 CFU / mL), 15-30mL spray treatment, preferably 20mL spray treatment; or, when controlling garlic leaf blight in the field, the application dosage of the microbial agent is: JSBV63 fermentation liquid (bacteria content is 10 8 CFU / mL), 1-6L / mu, preferably 2-4L / mu, and the fermentation liquid is diluted with 60L of water for foliar spraying.

[0023] Furthermore, when applying to the above-ground part of the plant, for example, a method of foliar spraying may be adopted; when applying to the growth environment of the plant root system, for example, a method of root irrigation or furrow application may be adopted.

[0024] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0025] The present invention isolates and screens a strain of Bacillus Velezii JSBV63 for the first time, which is isolated from soil and harmoniously blends with the ecological environment. It is non-toxic and non-pathogenic to humans, livestock and crops, has simple culture conditions, is easy to industrialize and has good application prospects. It belongs to a biological preparation, can reduce environmental pollution, avoids the problems caused by the use of chemical control preparations, and is beneficial to the pollution-free green production of garlic. The JSBV63 strain can significantly inhibit the growth of Stipes stolonifer, has a good inhibitory effect on it, and has a good control effect on garlic leaf blight caused by Stipes stolonifer, can effectively control the occurrence of garlic leaf blight, has a good disease prevention effect, and has a large potential to be developed into a commercial live bacterial biocontrol preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0027] Figure 1 is a colony morphology diagram of Bacillus Velez JSBV63 in one embodiment of the present invention;

[0028] Figure 2 Schematic diagram of the results of the inhibitory effect of Bacillus Velez JSBV63 on the mycelial growth of garlic leaf blight pathogen in one embodiment of the present invention; wherein the diameter of the culture dish is 9 cm;

[0029] Figure 3 This is a schematic diagram of the potted control effect of Bacillus Velez JSBV63 on garlic leaf blight in one embodiment of the present invention;

[0030] The above-mentioned Bacillus velezensis JSBV63 has been deposited, its deposit number is CGMCC No.24638, its classification name is Bacillus velezensis, the deposit date is April 6, 2022, the depositor is the General Microbiology Center of China Culture Collection Administration (CGMCC for short), and the depositor’s address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0031] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0032] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0033] Example 1 - Isolation, Screening and Identification of Bacillus Velezii JSBV63

[0034] This example is a screening and identification process of Bacillus velezensis JSBV63, which specifically includes the following steps:

[0035] (1) Isolation of strain JSBV63

[0036] The separation was performed by plate dilution method. The specific method includes: taking 1g of soil samples collected from the garlic production area of ​​Fengxian County, Jiangsu Province, and placing them in a sterile conical flask containing 27mL of 0.85% sodium chloride solution by mass volume, placing them on a shaker at 150rpm, shaking and incubating at 28℃ for 30min, and then letting them stand for 5min. The suspension at this time is considered to be 10 -1 The diluent was then diluted 10 times with 0.85% sodium chloride solution by mass volume to prepare 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Dilution, pipette 10 -4 , 10 -5 , 10 -6 0.1 ml of three gradient dilutions were spread on LB solid medium, with 3 replicates for each gradient, and cultured at 30°C for about 3 days. Single colonies with different colony morphology were selected and separated and purified by continuous streaking on LB medium. The purified strains were transferred to LB slant medium for culture and stored at 4°C for future use. A total of 72 bacterial strains were isolated.

[0037] (2) Observation of morphological characteristics of strain JSBV63

[0038] The strain JSBV63 was streaked onto LB medium and cultured at 37°C for 24 hours before observing the morphological characteristics of the strain. Figure 1 As shown, on LB solid medium, pale yellow opaque colonies were found with a smooth surface, wrinkled edges and a slight bulge in the middle.

[0039] (3) Molecular characterization of strain JSBV63

[0040] The total genomic DNA of strain JSBV63 was used as a template, and the 16S rRNA of strain JSBV63 was amplified by PCR with primer pair 27-F (5'-AGAGTTTGATCCTGGCTCAG-3') / 1492-R (5'-TACGGCTACCTTGTTACGACTT-3'). After PCR cleaning, recovery, ligation, transformation, identification, and positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain nucleotide sequence information. The recovery was performed using the DNA gel recovery kit of Axygen, and the ligation was performed using the DNA gel recovery kit of TransGen. -Blunt Cloning Kit, and submit the obtained sequence to the GenBank database for BLAST analysis and comparison.

[0041] The sequence of 16S rRNA is shown in SEQ ID No. 1, with a total length of 1001 bp, and its homology with Bacillus velezensis is between 99.50-99.60%. Combined with the morphological characteristics of the strain and with reference to Bergey's Manual of Systematic Bacteriology, the Bacillus strain JSBV63 was preliminarily identified as Bacillus velezensis, and the strain was deposited with the deposit number CGMCC No. 24638.

[0042]

[0043]

[0044] Example 2 - Cultivation of strain JSBV63 and preparation of its inoculum

[0045] This example is a preferred culture method and bacterial preparation method of Bacillus Velez JSBV63 obtained by screening in Example 1, and the specific steps include:

[0046] The Velezella JSBV63 was inoculated into LB medium for activation, and cultured in a 37°C constant temperature incubator in the dark for 24 hours. The reserved JSBV63 was inoculated into 1L LB liquid medium for fermentation, and the fermentation liquid (10 8 CFU / mL) and stored at 4°C for future use.

[0047] The LB medium or LB liquid medium is composed of 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar, and is diluted to 1000 ml with distilled water and sterilized at 121° C. for 20 min.

[0048] Example 3 - Plate antagonistic effect assay of strain JSBV63

[0049] This example verifies the plate antagonistic effect of Bacillus Velez JSBV63 on pathogenic bacteria, and the steps include:

[0050] The garlic leaf blight pathogen, Stemphylium solani (isolated by our laboratory from garlic leaf blight diseased plants in Fengxian County, Xuzhou City, Jiangsu Province, and stored in a 4°C refrigerator; it is understandable that the above-mentioned Stemphylium solani can also be a commercially available strain) was cultured on PDA solid medium. PDA solid medium consists of 200g of potatoes (peeled), 20g of glucose, and 15g of agar, which is diluted to 1000ml with distilled water and sterilized at 121°C for 20min. The pathogen was cultured on PDA solid medium for 7 days for use.

[0051] Use a sterilized puncher to punch holes along the edge of the prepared pathogen hyphae to obtain a 5mm diameter bacterial cake, and inoculate the obtained bacterial cake in the center of a new PDA plate. The treatment group used a sterile inoculation loop to inoculate the JSBV63 strain (biocontrol bacteria after 24 hours of dark culture) in four directions 25mm away from the bacterial cake. The treatment was set up with 3 replicates, cultured in the dark at 28℃ for 4-7 days, and the colony diameter was measured by the cross method. The inhibition rate was calculated using the following formula:

[0052] Inhibition rate (%) = (control pathogen colony diameter - treatment pathogen colony diameter) / control pathogen colony diameter × 100%

[0053] Table 1 Antibacterial activity of JSBV63 strain against garlic leaf blight pathogens

[0054]

[0055] The results of antibacterial activity were as follows Figure 2 As shown in Table 1, it shows that the strain JSBV63 has a significant inhibitory effect on garlic leaf blight pathogens. The inhibition rates of each group of tests are: 78.31%, 77.85%, 78.15%, and the average inhibition rate is 78.10%.

[0056] Example 4 - Effect of fermentation liquid of strain JSBV63 on garlic leaf blight in potted plants

[0057] This example verifies the potted control effect of Bacillus Velez JSBV63 on garlic leaf blight caused by Stemphylium solani, and the steps include:

[0058] The garlic cloves were sown in sterilized soil. After 45 days, the garlic seedlings were transplanted into pots filled with sterilized soil, with 3 plants per pot and 15 plants per treatment, and conventional management was performed. After the garlic leaf blight pathogen was cultured on PDA solid medium for 7 days, spores were scraped and made into spore suspension with sterile water. The spore concentration was 1×10 6 Each pot was inoculated by spraying 50 ml of the solution. One hour after inoculation, the JSBV63 fermentation liquid (with a bacterial content of 10 8 CFU / mL) 20mL spray treatment. Clean water treatment and blank (not inoculated with garlic leaf blight pathogen) were used as controls. The disease was investigated after 14 days and the control effect was calculated.

[0059] Disease grade determination standard: Grade 0: no disease; Grade 1: the area of ​​lesions accounts for less than 5% of the total leaf area; Grade 3: the area of ​​lesions accounts for less than 6% to 10% of the total leaf area; Grade 5: the area of ​​lesions accounts for less than 11% to 20% of the total leaf area; Grade 7: the area of ​​lesions accounts for less than 21% to 50% of the total leaf area; Grade 9: the area of ​​lesions accounts for more than 50% of the total leaf area. The control effect calculation method is as follows:

[0060]

[0061]

[0062] In the formula: CK is the control group, PT is the treatment group.

[0063] Table 2 Determination of potted control effect of strain JSBV63 fermentation liquid on garlic leaf blight

[0064]

[0065] The results are as follows Figure 3 As shown in Table 2, 14 days after inoculation with garlic leaf blight pathogen, the leaves of garlic plants in the control group showed typical leaf blight symptoms, and some plants fell over, with a disease index of 6.49. Plants treated with biocontrol bacteria JSBV63 showed less disease, with a disease index of only 1.19, and a potted plant control efficiency of 81.66%, which was significantly different from that of the water treatment.

[0066] Example 5 - Field control effect of the fermentation liquid of strain JSBV63 on garlic leaf blight

[0067] This example verifies the field control effect of Bacillus Velez JSBV63 on garlic leaf blight caused by Stemphylium solani, using the JSBV63 fermentation liquid prepared in Example 2, and the steps include:

[0068] The field plot test was conducted in the garlic field of Shouxian Town, Fengxian County, Xuzhou City, Jiangsu Province in 2019. A total of 5 treatments were set up, 4 replicates, and the plot area was 20 square meters. Treatment 1: JSBV63 fermentation liquid (4L / mu); Treatment 2: JSBV63 fermentation liquid (2L / mu); Treatment 3: difenoconazole (active ingredient dosage 20g / mu); Treatment 4: triadimefon (active ingredient dosage 20g / mu); Treatment 5: clear water control. The above treatment agents were diluted with 60L of water for foliar spraying.

[0069] The sampling method for the investigation of the disease control effect is carried out according to the following steps. Five points are sampled on the diagonal of each plot, with 4 plants at each point. All leaves of each plant are investigated, and the grades are graded according to the percentage of the disease spot area on each leaf to the entire leaf area. The disease grade determination standard is the same as in Example 4. The control effect is calculated (the formula is the same as in Example 4).

[0070] Table 3 Field control effect of JSBV63 fermentation liquid on garlic leaf blight

[0071]

[0072] The results are shown in Table 3. After 14 days of treatment with JSBV63 fermentation liquid (4L / mu), the control effect on garlic leaf blight reached 61.49%, which is equivalent to the control effect of the chemical agent difenoconazole. Compared with another chemical agent triadimefon, the control effect is significantly different. This shows that the fermentation liquid of strain JSBV63 can be used for field control of garlic leaf blight.

[0073] It can be seen from the above examples that Bacillus Velezii JSBV63 can effectively inhibit the growth of Stemphylium solani, and can effectively prevent and control garlic leaf blight caused by Stemphylium solani in potted plants and fields. This shows that the JSBV63 bacterial agent has a good effect in preventing and controlling garlic leaf blight and has great potential to be developed into a commercial live bacterial biocontrol agent.

[0074] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus velez Bacillus velezensis ), characterized in that, The name of the Bacillus Velez subtilis is JSBV63, and its deposit number is CGMCC No. 24638; wherein, the sequence of 16SrDNA of the Bacillus Velez subtilis JSBV63 is shown as SEQ ID No.

1.

2. A microbial agent, characterized in that: The microbial agent comprises at least one of the bacterial cells, spores and fermentation broth of Bacillus Velezii as claimed in claim 1.

3. The microbial agent according to claim 1, characterized in that: The live bacteria concentration of the microbial agent is 5×10 7 ~5×10 8 CFU / mL.

4. A microbial agent according to claim 2, characterized in that: The microbial agent is a fermentation broth of Bacillus Velez subtilis, and the preparation of the fermentation broth includes the steps of inoculating Bacillus Velez subtilis JSBV63 in LB culture medium, culturing it in the dark for a predetermined time, inoculating it in LB liquid culture medium for fermentation, and obtaining the fermentation broth of Bacillus Velez subtilis JSBV63 after culturing it for a predetermined time.

5. The microbial agent according to claim 4, characterized in that The dark culture conditions are: 30-37° C., culture for 12-36 h; and / or, the fermentation conditions are: fermentation temperature of 30-37° C., fermentation time of 48-72 h, and shaking culture at a rotation speed of 180-220 rpm.

6. A use of the Bacillus Velezii according to claim 1 or the microbial agent according to any one of claims 2 to 5, characterized in that: The application includes at least one of the following applications: application in inhibiting the growth of pathogenic bacteria, application in preventing and controlling plant diseases caused by pathogenic bacteria; wherein the pathogenic bacteria is Pseudomonas stolonifera ( Stemphylium solani ), the plant is a plant of the Liliaceae family.

7. The use according to claim 6, characterized in that: The plant disease is garlic leaf blight.

8. A method for inhibiting the growth of pathogenic bacteria of the genus Stipes or a method for preventing and controlling plant diseases caused by pathogenic bacteria of the genus Stipes, characterized in that: The method comprises the steps of: applying the Bacillus Velezii according to claim 1 or the microbial agent according to any one of claims 2 to 5 to the pathogen, the aerial part of the plant or the growth environment of the plant root system; wherein the pathogen of the genus Stipes is Stipes ( Stemphylium solani ).

9. The method according to claim 8, characterized in that The plant disease is garlic leaf blight.

10. The method according to claim 9, characterized in that When controlling garlic leaf blight in potted plants, the application dosage of the microbial agent is: 15-30 mL spray treatment; or, when controlling garlic leaf blight in the field, the application dosage of the microbial agent is 1-6 L / mu.

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