A strain of Bacillus tequilensis ZW-35 and its applications

Abstract

The present invention provides a Bacillus tequilensis ZW-35 and its application, which relates to the technical field of microbial control. The Bacillus tequilensis ZW-35 is preserved in the China General Microbiological Culture Collection Center, and its taxonomic name is Bacillus tequilensis Bacillus tequilensis , and the preservation number is CGMCC No. 32878. The volatile substances in the metabolic process of Bacillus tequilensis ZW-35 can effectively inhibit the growth of Colletotrichum; and the volatile substances have high diffusion ability in air and soil pores. In the prevention and control of fruit diseases, the volatile substances can avoid direct contact with the fruits and will not leave residues on the fruit surface, thus avoiding the potential risks brought by direct contact.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial control, and particularly to a strain of Bacillus tequilensis ZW-35 and its application. Background Art

[0002] Colletotrichum is a kind of fungus, belonging to Deuteromycotina, Coelomycetes, Melanconiales, Melanconiaceae, Colletotrichum. The morphology of Colletotrichum includes conidia, septate hyphae, acervulus, setae and other components. Its life cycle includes conidial stage and hyphal stage, and it enters the plant body by infecting plant wounds or natural orifices, causing plant tissue necrosis and rot.

[0003] After a plant is infected with Colletotrichum, yellow or white lesions will appear on leaves, stems and fruits. As the disease progresses, the lesions gradually expand, tissue necrosis occurs, and ultimately the plant dies. After a plant is infected with Colletotrichum, its photosynthesis ability decreases, the synthesis of nutrients decreases, thereby causing the plant to grow slowly and the yield to decrease. In addition, plant anthracnose may also lead to a decrease in the stress resistance of the plant, making it vulnerable to infection by other pathogens.

[0004] At present, the control of Colletotrichum mainly includes chemical control, physical control, agricultural control and biological control. Among them, chemical control mainly uses chemical agents to control the diseases caused by Colletotrichum in plants. Commonly used chemical agents include carbendazim, thiophanate-methyl, difenoconazole, etc. However, the use of chemical reagents is likely to cause pollution to plants and the environment; physical control mainly uses ultraviolet lamps to kill conidia on the surface of plants to reduce the number of pathogens, or uses high-temperature treatment. However, the timing requirements for plant treatment are relatively high, and the growth of plants may also be affected after the final treatment, and the application range is small; biological control uses microbial resources for control. Some microorganisms such as Bacillus and Trichoderma have the effect of inhibiting the growth of Colletotrichum in plants and can be used as candidate strains for biological control. Relatively speaking, biological control has the advantages of environmental protection and sustainability. However, more verification and determination of relevant microorganisms are needed for the effect requirements of biological control. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a strain of Bacillus tequilensis ZW-35 and its application. The volatile substances metabolized by Bacillus tequilensis ZW-35 can effectively inhibit the growth of Colletotrichum and control the deterioration and development of fruit anthracnose, providing an effective biological control for the prevention and control of Colletotrichum.

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] A strain of Bacillus tequilensis ( Bacillus tequilensisZW-35, wherein the Bacillus tequilensis ZW-35 is deposited in the China General Microbiological Culture Collection Center, and the taxonomic name is Bacillus tequilensis Bacillus tequilensis , and the deposit number is CGMCC No. 32878.

[0008] Bacillus tequilensis ZW-35 is applied to inhibit Colletotrichum.

[0009] Preferably, the application method is to inhibit Colletotrichum with the volatile substances of Bacillus tequilensis ZW-35.

[0010] Bacillus tequilensis ZW-35 is applied to control fruit anthracnose.

[0011] Preferably, the application method is to control fruit anthracnose with the volatile substances of Bacillus tequilensis ZW-35

[0012] The present invention provides a strain of Bacillus tequilensis ZW-35 and its application. Compared with the prior art, the advantages are as follows:

[0013] The present invention provides a strain of Bacillus tequilensis ZW-35, and the volatile substances in its metabolic process can effectively inhibit the growth of Colletotrichum; and the volatile substances have high diffusion ability in air and soil pores. In the prevention and control of fruit diseases, the volatile substances can not directly contact with the fruits, nor leave residues on the fruit surface, avoiding the potential risks brought by direct contact. Description of the Drawings

[0014] Figure 1 It is a schematic diagram of the colony morphology of Bacillus tequilensis ZW-35 of the present invention;

[0015] Figure 2 It is a phylogenetic tree of Bacillus tequilensis ZW-35 of the present invention;

[0016] Figure 3 It is a schematic diagram of the antibacterial effect of the petri dish at 8 d in Example 2 of the present invention. The antibacterial effect of the control petri dish is on the left, and the antibacterial effect of the petri dish treated with the volatile substances of Bacillus tequilensis is on the right;

[0017] Figure 4 It is a schematic diagram of the structure of the hyphal cells of Colletotrichum cultured in the blank control group for 8 d in Example 2 of the present invention;

[0018] Figure 5 It is a schematic diagram of the structure of the hyphal cells of Colletotrichum cultured in the treatment group with the volatile substances of Bacillus tequilensis for 8 d in Example 2 of the present invention;

[0019] Figure 6This is a schematic diagram of the lesion growth of mangoes placed for 12 days in Example 3 of the present invention. The left side is the sterile water treatment group, and the right side is the volatile substance treatment group of Bacillus tequilensis. Detailed implementation manners

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] The following Bacillus tequilensis ZW-35 strain is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The taxonomic name is Bacillus tequilensis Bacillus tequilensis , with the preservation number of CGMCC No. 32878 and the preservation date of December 02, 2024. Example 1:

[0022] Isolation and identification of Bacillus tequilensis ZW-35:

[0023] Bacillus tequilensis ZW-35 was isolated from healthy oil tea leaves:

[0024] The dilution plating method was used to isolate the endophytic bacteria of oil tea leaves. The specific operation is as follows: Place healthy oil tea leaves in a 75% ethanol solution and soak for 2 - 3 s. Then wash them 3 times with sterile water to remove the residual ethanol solution. Finally, cut the healthy tissue with a sterile scalpel and transfer it to an LB solid medium, and culture it in an incubator at 28°C for 48 h. Purify the strain by the streaking method ( Figure 1 ). Finally, identify the purified strain using 16S rRNA. The results show that the full length of the 16S rRNA sequence of Bacillus tequilensis ZW-35 is 1438 bp, and the sequence similarities with the strains Bacillus tequilensis Bacillus tequilensis SJ33 and Bacillus tequilensis Bacillus tequilensis 10b are 99.50% and 99.40% respectively (Table 1), and it is in the same phylogenetic branch as the strains of Bacillus tequilensis Bacillus tequilensis species ( Figure 2 ). Therefore, the strain ZW-35 was identified as Bacillus tequilensis Bacillus tequilensis .

[0025] Table 1

[0026] Example 2:

[0027] 1. Antibacterial effect of volatile substances of Bacillus tequilensis ZW-35 on Colletotrichum gloeosporioides:

[0028] Use a punch to cut out a 5-mm Colletotrichum gloeosporioides mycelial disc and inoculate it in the center of the left medium of the two-compartment medium. Coat 100 μL of a 10 7 CFU / mL Bacillus tequilensis suspension on the right side. Use the blank medium without inoculating Bacillus on the right side as a control. Each treatment is repeated 3 times and cultured at 28 °C for 8 d. Measure the colony diameter of the test pathogen and calculate the inhibition rate. The specific results are as Figure 3 and Table 2 below show:

[0029] Table 2

[0030]

[0031] As can be seen from the above table, the volatile substances of Bacillus tequilensis have a good inhibitory effect on Colletotrichum acutatum, and the inhibition rate is 55.6% after 8 days.

[0032] 2. Damage of volatile substances of Bacillus tequilensis ZW-35 to the hyphal cells of Colletotrichum gloeosporioides:

[0033] Collect the hyphae of Colletotrichum gloeosporioides by the method of overlay culture. Place a purified and cultured Colletotrichum gloeosporioides mycelial disc taken with a punch in the middle of the prepared PDA solid medium. Then, invert the LB solid medium coated with 200 μL of Bacillus tequilensis ZW-35 on the PDA solid medium with the Colletotrichum gloeosporioides mycelial disc. Seal it with a sealing film and culture it at 28 °C for 5 - 8 d. Use the non-inoculated LB medium as a blank control. After 8 d, use a scalpel to cut the hyphae of Colletotrichum acutatum in the control group and the treatment group, fix them with glutaraldehyde, and then observe them under a transmission electron microscope. Specifically, as shown in Figure 4 (blank control group) and Figure 5 (treatment group with volatile substances of Bacillus tequilensis).

[0034] The results show that the volatile substances of Bacillus tequilensis destroy the structural integrity of the hyphal cells of Colletotrichum gloeosporioides, damage the cell wall and cell membrane of the cells, and the cell contents leak out. Example 3:

[0035] Control of mango anthracnose by volatile substances of Bacillus tequilensis ZW-35:

[0036] Mango fruits without mechanical damage, of uniform size, free from pests and diseases, and with consistent maturity were selected as experimental materials, disinfected with sodium hypochlorite solution, washed with sterile water, and air-dried for later use. A sterilized pipette tip was used to puncture the fruits at the equatorial part (4 mm deep and 4 mm in diameter), and 10 μL of a spore suspension of Colletotrichum gloeosporioides with a concentration of 10 4 CFU / mL was quantitatively injected into the holes. Three fruits were used for each treatment, and the experiment was repeated three times. Then the mangoes were placed in plastic boxes. An LB solid medium coated with 200 μL of a Bacillus tequilensis ZW-35 bacterial suspension with a concentration of 10 7 CFU / mL was placed in the middle of the box containing the mangoes inoculated with the pathogen after opening the lid, and then the lid of the plastic box was covered. A Petri dish without the inoculated Bacillus was used as a control. The incidence and lesion conditions of the fruits were observed every day. The growth of the lesions at 12 days is shown in Figure 6 Figure; The incidence calculation formula is as follows: Incidence (%) = (number of diseased holes / total number of holes) × 100%. Each treatment was repeated 3 times, and the specific incidences are shown in Table 3 below:

[0037] Table 3

[0038]

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Bacillus tequila ( Bacillus tequilensis )ZW-35, characterized by: The tequila bacillus ZW-35 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration and is classified and named as tequila bacillus ( Bacillus tequilensis ), the deposit number is CGMCC No.32878.

2. Use of the Bacillus tequila ZW-35 as claimed in claim 1 in inhibiting mango anthracnose.

3. The Bacillus tequila ZW-35 as claimed in claim 1 is used for preventing and controlling mango anthracnose.

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