A biocontrol bacteria strain Bacillus subtilis deserticola ZG8 and its application

Abstract

The invention discloses a biocontrol bacteria strain Bacillus subtilis desert subspecies ZG8 and its application, belonging to the technical field of plant disease prevention and control, and a biocontrol bacteria strain Bacillus subtilis desert subspecies ZG8, wherein the biocontrol strain is Bacillus subtilis desert subspecies ZG8, which is deposited in the China General Microbial Culture Collection Center CGMCC, with a deposit number of CGMCC NO.31897. The biocontrol strain in the invention is applied to plant disease prevention and control, and the plant diseases include crop diseases caused by Fusarium pseudosporum, Fusarium graminearum, Botrytis cinerea, Alternaria alternata, Rhizoctonia solani and Pseudomonas aeruginosa. The control effect on tobacco brown spot disease on leaves is 63.55%, and the control effect on damping-off disease on tobacco rhizomes in potted experiments is 55.17%. The invention can effectively prevent and control tobacco diseases, significantly reduce the incidence of tobacco, and promote the growth of tobacco seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease prevention and control, and specifically relates to a biocontrol bacteria strain Bacillus subtilis deserticola ZG8 and an application thereof. Background Art

[0002] As a key cash crop in my country, tobacco faces many challenges in its production process. With the continuous changes in climate conditions and cultivation techniques, tobacco is facing increasingly severe disease problems, especially the incidence of leaf diseases, which are increasing year by year. Leaf diseases include Fusarium root rot, brown star disease, damping-off, target spot disease and gray mold, which have a huge impact on flue-cured tobacco production. These diseases not only cause leaf damage and plant death, but also reduce the quality and yield of tobacco leaves, ultimately affecting the overall benefits of the tobacco industry.

[0003] Traditional agricultural control methods face many limitations such as high cost and difficulty in implementation. Although the use of chemical pesticides can effectively control plant diseases, it is often accompanied by a series of problems such as pesticide residues, increased pathogen resistance, ecological damage, safety risks and sustainability challenges. Moreover, chemical pesticides not only have long-term adverse effects on human health and the environment, but may also accelerate the development of pathogen resistance, disrupt the ecological balance, and pose a direct threat to users if improperly operated. To this end, a biocontrol strain Bacillus subtilis desert subsp. ZG8 and its application were proposed.

[0004] The above information disclosed in this background technology is only used to increase the understanding of the background technology of the present invention and therefore, it may include information that does not constitute the prior art known to ordinary technicians in this field. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one object of the present invention is to provide a biocontrol bacteria strain Bacillus subtilis deserticola ZG8 and its application. The biocontrol bacteria strain is used in the prevention and control of tobacco diseases, and plant diseases include diseases caused by Fusarium sporotrichioides, Fusarium graminearum, Botrytis cinerea, Alternaria alternata, Rhizoctonia solani and Pseudomonas citrinopileatus.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A biocontrol bacteria strain Bacillus subtilis deserticola ZG8 is disclosed. The biocontrol bacteria strain is Bacillus subtilis deserticola ZG8, which is deposited in the China General Microbiological Culture Collection Center CGMCC, with a deposit number of CGMCC NO.31897.

[0008] The method for culturing Bacillus subtilis deserticola ZG8 is applied to biocontrol strains. The biocontrol strains are inoculated into LB medium for culturing at a temperature of 37°C.

[0009] The application of a biocontrol strain Bacillus subtilis subspecies desert ZG8 against Fusarium sporotrichioides, Fusarium graminearum, Botrytis cinerea, Alternaria alternata, Rhizoctonia solani and Pseudomonas citrinopileatus.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The biocontrol bacteria strain ZG8 of the present invention can effectively inhibit a variety of tobacco disease pathogens, including Fusarium sporotrichioides, Fusarium graminearum, Botrytis cinerea, Alternaria alternata, Rhizoctonia solani and Pseudomonas aeruginosa. The control effect of strain ZG8 on tobacco brown spot disease is as high as 63.55%, and the control effect on damping-off disease on tobacco rhizomes in potted experiments is 55.17%. At the same time, strain ZG8 can also promote the growth of tobacco seedlings.

[0012] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a colony diagram of the biocontrol strain ZG8 of the present invention.

[0014] Figure 2 The figure is a plate confrontation effect diagram of the biocontrol strain ZG8 of the present invention and various pathogens; wherein A: Fusarium spp.; B: Fusarium graminearum; C: Botrytis cinerea; D: Alternaria alternata; E: Rhizoctonia solani; and F: Trichoderma cucurbitae.

[0015] Figure 3 This is a result diagram of the brown star disease spot area on detached leaves under different treatments of the present invention.

[0016] Figure 4 This is a result diagram of the agronomic traits of tobacco seedlings under different treatments of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the 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 of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; unless otherwise specified, the reagents or products involved are all commercially available conventional reagents or products; unless otherwise specified, the test methods involved are all conventional methods.

[0019] Embodiment 1

[0020] Isolation and identification of biocontrol strains

[0021] (1) Isolation of biocontrol strains: The biocontrol strains were isolated from tomato rhizosphere soil samples from fumigated soil in Jimo District, Qingdao City, Shandong Province, and cultured in LB medium at 37°C.

[0022] (2) Identification of biocontrol strains: The strain obtained in (1) was inoculated on an LB plate and cultured at 37°C for 2 days. Figure 1 As shown, the colonies are nearly circular and irregular, light yellow-brown, opaque, with jagged edges and slightly poor gloss.

[0023] The 16S rDNA sequence of the strain was determined using bacterial universal primers. The total DNA of strain ZG8 was used as a template to amplify the 16S rDNA fragment of the strain by PCR under the guidance of primer pair 27F (5'-AGAGTTTGATCCTGGCTCAG-3') / 1492R (5'-ACGGCTACCTTGTTACGACT-3'). The PCR reaction system was: 2×PowerTaq PCR MasterMix: 10μL, template DNA: 1μL, upstream primer (10μmol / L): 1μL, downstream primer (10μmol / L): 1μL, ddH2O: 12μL, total volume: 25μL. PCR reaction program: after 95℃ pre-denaturation for 3min, enter the following cycle of 94℃ denaturation for 15s, 55℃ annealing for 15s, 72℃ extension for 15s, 72℃ extension for 5min after 34 cycles, and finally stored in the PCR instrument at 4℃.

[0024] The PCR product was sequenced by Qingdao Weilai Biotechnology Co., Ltd., and the sequence was submitted to the NCBI database for homology comparison by BLAST. The results showed that there was 100% homology with Bacillus subtilis subsp. inaquosorum.

[0025] The Bacillus subtilis desert subspecies strain screened by the present invention was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China) on September 6, 2024, and its biological deposit number is CGMCC NO.31897.

[0026] Embodiment 2

[0027] The confrontation culture method was used, and a hole with a diameter of 8 mm was used to punch holes in the activated pathogen plate, and the bacterial block was inoculated in the center of the PDA plate.

[0028] The biocontrol strains were spotted at 2 cm around the pathogen block, and the plate inoculated with only pathogens was used as a control. After culturing at 28°C for 7 days, the antibacterial ability of the biocontrol strains was measured.

[0029] The results are shown in Table 1 and Figure 2 As shown, the biocontrol strain has a strong inhibitory effect on Fusarium graminearum, Fusarium graminearum, Botrytis cinerea, Alternaria alternata, Rhizoctonia solani and Corynebacterium cucurbitae.

[0030]

[0031] Table 1 Inhibition rate of biocontrol strain ZG8 against different pathogens

[0032] Embodiment 3

[0033] The specific steps of the test on the efficacy of biocontrol strains on tobacco leaf brown spot disease are as follows:

[0034] Step 1: Activate the red star pathogen on PDA medium, culture the biocontrol strain in LB medium for 48 hours, centrifuge, resuspend with sterile water, and adjust the bacterial solution concentration to OD 600 =1.0.

[0035] Step 2: Spray the obtained bacterial suspension evenly onto 4-6 week old Nicotiana benthamiana, using sterile water as a control, and then use a 5 mm diameter punch to punch the pathogen into bacterial cakes.

[0036] Step 3: Use the needle tip of a 1mL sterile syringe to create trauma on the tobacco leaves, place the pathogen on the back of the tobacco leaves, use agar blocks as a control, record the incidence after 48 hours, and use Image-J software to count the diseased area.

[0037] The results are as follows Figure 3 As shown in Table 2, the control effect of strain ZG8 on brown spot disease on leaves was as high as 63.55%.

[0038]

[0039] Table 2 Effect of biocontrol strain ZG8 on control of brown spot disease

[0040] Embodiment 4

[0041] The specific steps for testing the potted plant efficacy of biocontrol strains against tobacco damping-off disease are as follows:

[0042] Step 1: Activate the bacterium Rhizoctonia solani on PDA medium, culture the biocontrol strain in LB medium for 48 hours, centrifuge, resuspend with sterile water, and adjust the bacterial solution concentration to OD 600 =1.0.

[0043] Step 2: Use the tip of a 1mL sterile syringe to create a wound at the base of the stem of Nicotiana benthamiana, draw 1mL of bacterial suspension into the area, use sterile water as a control, then use a 5mm diameter puncher to punch the pathogen into a bacterial cake and place it on the wound, set up 3 replicates, each with 5 strains.

[0044] Step 3: After moisturizing with humidified cotton balls for 48 hours, remove the bacterial cake and cotton balls, place them at room temperature, and record the incidence after 7 days.

[0045] The classification standard of tobacco damping-off disease refers to the classification standard of tobacco black shank disease (National Standard of the People's Republic of China GB / T 23222-2008). The classification survey is conducted on the basis of the whole plant.

[0046] Level 0: The whole plant is disease-free.

[0047] Level 1: The stem lesions do not exceed 1 / 3 of the stem circumference, or less than 1 / 3 of the leaves are wilted.

[0048] Level 3: The lesions on the stem surround 1 / 3 to 1 / 2 of the stem circumference, or lesions appear on a few leaves at the bottom, or 1 / 3 to 1 / 2 of the leaves are slightly wilted.

[0049] Level 5: The stem lesions cover more than 1 / 2 of the stem circumference but do not reach the entire stem circumference, or 1 / 2 to 2 / 3 of the leaves are wilted.

[0050] Level 7: The stem lesions surround the entire stem, or more than 2 / 3 of the leaves wilt.

[0051] Level 9: The diseased plants are basically dead.

[0052] Specifically,

[0053] The results are shown in Table 3. The control effect of strain ZG8 on tobacco damping-off disease in the pot experiment was 42.42%.

[0054]

[0055] Table 3 Effect of biocontrol strain ZG8 on controlling damping-off disease

[0056] Embodiment 5

[0057] The specific steps of the test on promoting the growth of tobacco seedlings by biocontrol strains are as follows:

[0058] Step 1: The biocontrol strain ZG8 was cultured in LB medium for 48 h, then centrifuged and resuspended in sterile water.

[0059] Step 2: Adjust the bacterial concentration to OD 600 =1.0, the tobacco seedlings at the big cross stage were treated by root irrigation, with sterile water as the control, 6 replicates were set for each treatment, and the growth of the tobacco seedlings was counted after one month.

[0060] The results are as follows Figure 4 As shown, compared with the control, strain ZG8 significantly promoted the aboveground fresh weight, plant height and maximum leaf area of ​​tobacco seedlings, but had no significant effect on the stem diameter of tobacco seedlings.

[0061] Parts not involved in the present invention are the same as the prior art or can be implemented by the prior art. Although the embodiments of the present invention have been shown and described, it is understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biocontrol bacteria strain Bacillus subtilis deserticola ZG8, characterized in that: The biocontrol strain is Bacillus subtilis deserticola ZG8, which is deposited in the China General Microbiological Culture Collection Center CGMCC, with a deposit number of CGMCCNO.31897.

2. Application of the biocontrol bacteria strain Bacillus subtilis deserticola ZG8 according to claim 1 in tobacco disease pathogens.

3. The use according to claim 2 comprises Fusarium sporotrichioides, Fusarium graminearum, Botrytis cinerea, Alternaria alternata, Rhizoctonia solani and Corynebacterium cucurbitae.

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