Bacillus pumilus, fungicide, method and application

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus pumilus, a microbial agent, a method and application. The invention provides a bacillus pumilus G15, and the preservation number of the bacillus pumilus G15 is CGMCC (China General Microbiological Culture Collection Center) No.32839. The bacillus pumilus G15 has a remarkable inhibition effect on pectobacterium variabilis FN20111, the invention further provides a fungicide, and potting control effect determination shows that the bacillus pumilus G15 has a high control effect on the pectobacterium variabilis FN20111.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus pumilus, a bacterial agent, a method and an application. Background Art

[0002] Potato blackleg is caused by Pectobacterium spp. Potato blackleg is a common bacterial disease in potato production, while aerial stem rot is a new disease caused by three new species of the same genus as the pathogen that causes blackleg, namely Pectobacterium parvum, Pectobacterium versatile, and Pectobacterium brasiliense.

[0003] Potato aerial stem rot is a bacterial disease. It mainly occurs in varieties with strong growth, such as Xisen 6, 226, fertile soil 5, and V7 under certain conditions. The disease is mainly caused by bacterial infection after the base of the stem is damaged, which causes black and sticky symptoms. If prevention and control are not timely, it will cause large-scale infection in the whole field, which has a great impact on the yield and quality of potatoes. The difficulty of preventing and controlling aerial stem rot is higher than that of late blight. Since aerial stem rot has only become popular in recent years, the disease pattern, whether the seed potatoes are infected, and efficient prevention and control methods are all imperfect. There is no strain with good prevention effect on potato aerial stem rot and potato black leg disease in the prior art. Summary of the invention

[0004] In view of this, the present invention provides a Bacillus pumilus, a bacterial agent, a method and an application. The Bacillus pumilus provided by the present invention can prevent and control potato aerial stem rot and potato black shank disease.

[0005] In order to solve the above technical problems, the present invention proposes the following technical solutions:

[0006] The invention provides a strain of Bacillus pumilus G15, whose deposit number is CGMCC No.32839.

[0007] The present invention provides a bacterial agent, comprising the Bacillus pumilus G15 described in the above technical solution.

[0008] Preferably, the effective viable count of Bacillus pumilus G15 in the bacterial agent is ≥ 1×10 7 CFU / mL.

[0009] Preferably, the application form of the Bacillus pumilus G15 includes bacterial suspension and / or fermentation broth.

[0010] Preferably, the preparation of the fermentation broth comprises: inoculating the Bacillus pumilus G15 into a culture medium for culturing to obtain a fermentation broth;

[0011] The preparation of the bacterial suspension includes: centrifuging and resuspending the fermentation liquid to obtain the bacterial suspension.

[0012] The present invention provides use of the Bacillus pumilus G15 described in the above technical solution or the bacterial agent described in the above technical solution in preventing and controlling pathogenic bacteria of the genus Pectobacterium (Pectobacterium spp.).

[0013] The present invention provides application of the Bacillus pumilus G15 described in the above technical solution or the bacterial agent described in the above technical solution in preventing and controlling potato diseases.

[0014] Preferably, the diseases include potato blackleg and / or potato aerial stem rot.

[0015] The present invention provides a method for preventing and controlling potato diseases, wherein the Bacillus pumilus G15 or the bacterial agent described in the above technical solution is applied to potatoes.

[0016] Preferably, when used in the field, the amount of the bacterial agent sprayed is 1.9-2.1 L / 667 m 2 When used for potted plants, the amount of the bacterial agent sprayed is 28 to 32 mL per seedling.

[0017] The invention has the beneficial effects as follows: the invention provides a strain of Bacillus pumilus G15, which has a significant inhibitory effect on Bacillus pumilus FN20111. The weight of the rotten part of the potato after G15 prevents and controls FN20111 is 0.23±0.04g, and the weight of the rotten part is reduced. The invention also provides a bacterial agent, and the potted plant control effect test shows that the control effect of Bacillus pumilus G15 on Bacillus pumilus FN20111 is 52.87%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The colony morphology and Gram staining of Bacillus pumilus G15;

[0019] Figure 2 The results of the screening of biocontrol strains of Bacillus pumilus to inhibit aerial stem rot pathogens are shown in Figure 1. The left side shows the inhibitory effect of G15 on Bacillus multiflorus FN20111, and the right side shows the water control treatment group.

[0020] Figure 3 This is the phylogenetic tree of closely related species of Bacillus brevis G15 constructed based on the maximum likelihood method;

[0021] Figure 4Phylogenetic trees constructed for strains FN20211 and FN20111 based on gapA gene sequences;

[0022] Figure 5 The toxicity of Pectinobacterium versatile and Bacillus pumilus G15 on potato tubers, wherein A is a longitudinal section of tubers inoculated with Pectinobacterium versatile (P.versatile) FN20111 and Bacillus pumilus G15; B is an analysis of the difference in the internal rot weight of tubers treated with different treatments;

[0023] Figure 6 The figure shows the effect of Bacillus pumilus G15 on the prevention of Pectinobacterium versatile FN20111 on potato tubers, where A shows the effect of strain G15 on FN20111 (P.versatile) on in vitro tubers; B shows the difference analysis of the internal rot weight of tubers with different treatments; P.versatile represents strain FN20111;

[0024] Figure 7 This is a potted plant protection effect diagram of Bacillus pumilus G15 against Pectinobacterium versatile (P.versatile) FN20111, where A is a longitudinal section of potato stems under different treatments; B is a difference analysis of the length of internal rot in the stems under different treatments; P.versatile represents strain FN20111.

[0025] Biological Deposit Description

[0026] Bacillus pumilus G15 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on November 28, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32839. DETAILED DESCRIPTION

[0027] The invention provides a strain of Bacillus pumilus G15, whose deposit number is CGMCC No.32839.

[0028] The Bacillus pumilus G15 of the present invention is screened by using rhizosphere soil samples as samples. The colonies of the Bacillus pumilus G15 on the LB culture medium plate are milky white, uniformly colored, and free of protrusions. The gyrB gene sequence is shown in SEQ ID No. 1, and the strain G15 is determined to be Bacillus pumilus based on the maximum likelihood method.

[0029] SEQ ID No.1:ATGCGTTATCTACGACYTTAGACGTGACCGTATATCGTG。

[0030] The potato tuber experiment and potted plant experiment in the embodiments of the present invention demonstrated that Bacillus pumilus G15 had a significant inhibitory effect on Pectinobacterium diversicolor FN20111, and was able to prevent and control potato aerial stem rot and potato black shank disease.

[0031] The present invention provides a bacterial agent, comprising the Bacillus pumilus G15 described in the above technical solution.

[0032] In the present invention, the effective viable count of Bacillus pumilus G15 in the bacterial agent is preferably ≥ 1×10 7 CFU / mL, more preferably 1×10 7 CFU / mL.

[0033] In the present invention, the application form of the Bacillus pumilus G15 preferably includes a resuspension and / or a fermentation broth.

[0034] In the present invention, the preparation of the fermentation broth preferably comprises: inoculating the Bacillus pumilus G15 into a culture medium for culturing to obtain a fermentation broth.

[0035] In the present invention, the Bacillus pumilus G15 is inoculated in a solid culture medium for activation culture to obtain a single colony of G15; the inoculation method of the present invention is preferably streak inoculation. There is no special limitation on the parameters of the streak inoculation, and conventional methods can be used. The temperature of the activation culture of the present invention is preferably 30°C to 37°C, and more preferably 33 to 37°C; in a specific embodiment of the present invention, the temperature of the activation culture is preferably 30, 32, 33, 34, 35, 36 or 37°C. The time of the activation culture is preferably 21 to 26h, and more preferably 23 to 24h; in a specific embodiment of the present invention, the time of the activation culture is preferably 21, 22, 23, 24, 25 or 26h. The activation culture is preferably completed under static conditions. The solid culture medium of the present invention preferably includes LB solid culture medium.

[0036] In the present invention, the obtained G15 single colony is preferably inoculated into a liquid culture medium for seed culture to obtain a seed solution. The present invention does not specifically limit the method of inoculation, and a conventional method can be used. The temperature of the seed culture of the present invention is preferably 33°C to 38°C, and more preferably 36 to 37°C; in a specific embodiment of the present invention, the temperature of the seed culture is preferably 33, 34, 35, 36, 37 or 38°C. The time of the seed culture of the present invention is preferably 12 to 19h, and more preferably 12 to 16h; in a specific embodiment of the present invention, the time of the seed culture is preferably 12, 13, 14, 15, 16, 17, 18 or 19h. The rotation speed of the seed culture is preferably 170 to 220rpm, and more preferably 180 to 200rpm. The liquid culture medium of the present invention preferably includes LB liquid culture medium.

[0037] In the present invention, the seed liquid is preferably transferred to a liquid culture medium for fermentation culture to obtain a fermentation liquid. The fermentation culture temperature of the present invention is preferably 31°C to 37°C, more preferably 35 to 37°C; in a specific embodiment of the present invention, the fermentation culture temperature is 31, 34, 35, 36 or 37°C. The volume of the seed liquid transferred is preferably 1% to 7% of the volume of the culture medium, more preferably 2% to 5%. The present invention preferably ferments and cultures until the OD of the culture liquid is 600 The rotation speed of the culture in the present invention is preferably 190 to 210 rpm, more preferably 195 to 200 rpm.

[0038] In the present invention, the preparation of the resuspension preferably comprises: centrifuging and resuspending the fermentation broth to obtain a bacterial suspension.

[0039] The present invention preferably centrifuges the fermentation broth to obtain G15 cells, washes the G15 cells, and then resuspends them to obtain a resuspension. The solution used for washing and resuspension in the present invention is preferably a 10mM NaCl solution or physiological saline. The present invention does not specifically limit the number of washing times, and conventional methods can be used.

[0040] The bacterial agent and Bacillus pumilus G15 of the present invention can be used alone or in combination with other Bacillus.

[0041] The present invention provides use of the Bacillus pumilus G15 described in the above technical solution or the bacterial agent described in the above technical solution in preventing and controlling pathogenic bacteria of the genus Pectobacterium (Pectobacterium spp.).

[0042] The Pectobacterium spp. pathogenic bacteria of the present invention preferably include Pectobacterium versatile.

[0043] The present invention provides application of the Bacillus pumilus G15 described in the above technical solution or the bacterial agent described in the above technical solution in preventing and controlling potato diseases.

[0044] The pathogenic bacteria of the disease of the present invention preferably include Pectobacterium spp. The Pectobacterium spp. of the present invention preferably include Pectobacterium versatile.

[0045] The diseases of the present invention preferably include potato black shank and / or potato aerial stem rot. Pectobacterium versatile is the pathogen of potato black shank and potato aerial stem rot, and Bacillus brevis G15 can prevent and control Pectobacterium spp. pathogens, which means that potato black shank and potato aerial stem rot can be prevented and controlled.

[0046] The application method of the present invention preferably comprises: applying the Bacillus pumilus G15 described in the above technical solution or the bacterial agent described in the above technical solution to potatoes.

[0047] The present invention provides a method for preventing and controlling potato diseases, wherein the Bacillus pumilus G15 described in the above technical solution or the bacterial agent described in the above technical solution is applied to potatoes.

[0048] The microbial agent of the present invention can be applied in the field or potted plants. The application amount in the field and potted plants is as follows: when used in the field, the application method preferably includes spraying; the application amount of the microbial agent spraying is preferably 1.9-2.1 L / 667 m 2 , more preferably 2L / 667m 2 The application time is preferably during the budding stage of potato plants, the application times are preferably 6 times, the first application time is preferably during the budding stage of potato plants, and the interval between two applications is preferably 10 days.

[0049] When used in potted plants, the application amount of the bacterial agent of the present invention is preferably 28-32 mL per potato plant, more preferably 30 mL; the application method of the present invention preferably includes spraying, the spraying site preferably includes the potato stem, the number of applications is preferably 1, and the spraying time is preferably about 30 days after the potato pot seedlings emerge.

[0050] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] Example 1 Isolation and purification of Bacillus strains in rhizosphere soil

[0052] Using the soil dilution method, 10 g of rhizosphere soil was weighed and added to a conical flask containing 90 mL of sterile water. The soil suspension was then aspirated after shaking at 200 rpm / min at 28°C for 30 min. 10 mL of the soil suspension was added to 90 mL of 85% saline and then diluted to 10% in a gradient manner after a water bath at 80°C for 15 min. -3 , 10 -4 , 10 -5 100 μL of each gradient was applied to a blank LB medium. Three replicates were set for each gradient treatment. The culture was then placed in a 37°C incubator for 24 hours. Single colonies were picked and streaked for purification to obtain multiple strains. The colony with wrinkled surface was recorded as strain G15.

[0053] A single colony of strain G15 without foreign bacteria was made into a seed solution and stored in a -80°C refrigerator with glycerol in a volume ratio of 1:1, and the mass concentration of glycerol was 50%.

[0054] The strain was streaked on a blank LB medium and the colony color, edge and morphological characteristics were observed under an optical microscope. The bacterial culture was stained with Gram stain and the color and shape of the bacteria were observed under an optical microscope. Figure 1 . Figure 1 This is the colony morphology and staining image of Bacillus pumilus G15.

[0055] Strain G15 appears milky white on the LB plate, with uniform coloration and no protrusions (see Figure 1 ), Gram staining was positive bacteria.

[0056] Example 2

[0057] 1. Materials and Methods

[0058] (1) Test strains

[0059] The potato aerial stem rot pathogens were provided by the Potato Disease and Plant Pathogen Innovation Team of the College of Plant Protection, Hebei Agricultural University. The potato aerial stem rot pathogens are Pectobacterium parvum FN20211 and Pectobacterium versatile FN20111; Pectobacterium parvum FN20211 can be found in “Wang Jinhui, Han Wanxin, Pirhonen Minna, Pan Yang, Zhang Dai, Zhao Dongmei, Li Qian, Cheng Jianing, Yang Zhihui, Zhu Jiehua. High-Quality Complete Genome Resource of Pectobacterium parvum Isolate FN20211 Causing Aerial Stem Rot of Potato. [J]. Molecular plant-microbe interactions: MPMI, 2022, 35(6): MPMI01220009A-MPMI01220009A.”;

[0060] Pectobacterium versatile (P.versatile) FN20111 see "Han Wanxin, Wang Jinhui, Li Zheng, Pan Yang, Zhang Dai, Zhao Dongmei, Li Qian, Yang Zhihui, Zhu Jiehua. First Report of Pectobacterium versatile Causing Aerial Stem Rot of Potato in China. [J]. Plant disease, 2021.".

[0061] (2) Test culture medium

[0062] The activation, preservation and propagation of pathogenic bacteria and Bacillus are carried out in Luria-Bertani (LB) medium. The specific formula of LB liquid medium is: 10g peptone, 5g yeast extract powder, 10g sodium chloride, distilled water to 1L, pH value adjusted to 7.0, and 20g agar powder per liter of LB solid medium.

[0063] 2. Screening of biocontrol strains - plate inhibition test

[0064] Pectobacterium microsus FN20211 and Pectobacterium diversicolor FN20111 were activated on LB medium plates, and single colonies were picked into LB liquid medium and cultured at 28°C and 200 rpm until OD 600 value = 0.8, and the culture solution of Pectinobacterium microsus FN20211 (referred to as culture solution 1) and the culture solution of Pectinobacterium diversum FN20111 (referred to as culture solution 2) were obtained respectively.

[0065] After strain G15 was activated, a single colony was picked and placed in LB liquid medium and cultured at 37°C and 200 r / min until OD 600 Value = 1.0, and the bacterial solution of strain G15 was obtained.

[0066] Initial screening process: Take 50 μL of culture solution 1 and spread it evenly on the LB medium plate. After standing for 5 minutes, place 5 filter papers. Pipette 10 μL of Bacillus pumilus G15 and drop it on the filter paper. After it dries, invert and culture it at 37℃ for 1 to 2 days and observe the results.

[0067] Set up a water control group-1: take 50 μL of culture solution 1 and spread it evenly on the LB medium plate. After standing for 5 minutes, place 5 filter papers. Pipette 10 μL of water and drop it on the filter papers. After it dries, invert and culture at 37℃ for 1 to 2 days, and then observe the results.

[0068] Take 50 μL of culture solution 2 and spread it evenly on the LB medium plate. After letting it stand for 5 minutes, place 5 filter papers. Pipette 10 μL of Bacillus pumilus G15 and drop it on the filter paper. After it dries, invert and culture it at 37°C for 1 to 2 days and observe the results.

[0069] Set up a water control group-2: Take 50 μL of culture solution 2 and spread it evenly on the LB medium plate. After standing for 5 minutes, place 5 filter papers. Pipette 10 μL of water and drop it on the filter papers. After it dries, invert and culture at 37℃ for 1 to 2 days, and then observe the results.

[0070] Rescreening process: Same as the initial screening process, the only difference is that instead of placing 5 filter papers in each dish, 1 filter paper is placed in the center of each dish. The rescreening results are shown in Figure 2 .

[0071] Through the plate antibacterial test, it was found that other strains isolated in Example 1, such as actinomycetes, Trichoderma and Pseudomonas, had no antagonistic effect on Pectobacterium, and only strain G15 had an antibacterial effect. Figure 2 It can be seen that strain G15 has a better inhibitory effect on diverse pectin Bacillus FN20111, with an inhibition zone diameter of 19.1±0.9 mm (see Figure 2 ). Strain G15 had no inhibitory effect on P. parvum FN20211.

[0072] Figure 2 Middle: The left side shows the inhibitory effect of G15 on Bacillus multiflorus FN20111; the right side shows the clear water control treatment.

[0073] 3. Molecular biological identification of biocontrol strains

[0074] (1) Strain G15 was identified as Bacillus pumilus (B. pumilus for short).

[0075] The specific identification process is as follows: the present invention downloads the genome information of 12 strains of Bacillus that have been sequenced from the NCBI database and performs gene similarity analysis on them.

[0076] Average nucleotide identity (ANI) refers to the similarity of homologous genes between two genomes. It is generally believed that when the ANI value is above 95%, they are the same species. The ANI value of strain G15 and Bacillus pumilus is as high as 99.49%, and the ANI values ​​of strain G15 and several other strains of Bacillus pumilus are all greater than 95%; the ANI values ​​of strain G15 and several other closely related Bacillus are all less than 95%. A DDH value of 70% is generally considered to be the critical value for different species. The DDH values ​​between strain G15 and several other strains of Bacillus pumilus are all higher than 70%, indicating that strain G15 is most closely related to Bacillus pumilus. At the same time, the whole genome sequence of the representative strain of each Bacillus was selected to construct a phylogenetic tree using iqtree, and strain G15 and B. pumilus were clustered into one branch. In summary, strain G15 was identified as Bacillus pumilus, as shown in Tables 1 and Figure 3 .

[0077] Table 1 Average nucleotide identity (ANI) and DNA homology (DDH) values ​​of closely related species of Bacillus

[0078]

[0079]

[0080] (2) Molecular biological identification of Pectinobacterium

[0081] According to the alignment of the sequencing results of the universal primers gapA-F and gapA-R of Pectinobacterium in NCBI, a phylogenetic tree was constructed ( Figure 4 ), and identified that FN20211 belonged to Pectobacterium parvum (P. parvum) and FN20111 belonged to Pectobacterium versatile (P. versatile). Figure 4This is a phylogenetic tree constructed based on the gapA gene sequences of strains FN20211 and FN20111.

[0082] 4. Determination of the efficacy of biocontrol strains against aerial stem rot

[0083] (1) Determination of pathogenicity of Pectobacterium and Bacillus on potato tubers

[0084] In order to clarify the toxicity of two pectin bacteria and the Bacillus that has an inhibitory effect on them on potato tubers, diverse pectin bacteria FN20111 and Bacillus pumilus G15 were selected to determine the pathogenicity on potato tubers. The specific process is as follows:

[0085] After streaking and activating Pectinobacterium versatile (P.versatile) FN20111 on an LB medium plate, the plate was cultured in a constant temperature incubator at 28°C for 24 hours to obtain a single colony of Pectinobacterium versatile FN20111. The obtained single colony of Pectinobacterium versatile FN20111 was picked and cultured in LB liquid medium at 28°C and 200rpm with shaking for 12 to 18 hours. The obtained culture solution was centrifuged at 8500rpm for 1 minute to collect the bacteria. The bacteria were washed with 10mM NaCl and resuspended to obtain a resuspension of Pectinobacterium versatile FN20111 (referred to as FN20111-resuspension 1).

[0086] After Bacillus pumilus G15 was streaked and activated on an LB medium plate, it was cultured in a constant temperature incubator at 37°C for 24 hours to obtain a single colony of Bacillus pumilus G15. The obtained single colony of Bacillus pumilus G15 was picked and cultured in LB liquid medium at 37°C and 200rpm with shaking for 12 to 18 hours. The obtained culture solution was centrifuged at 8500rpm for 1min to collect the bacteria. The bacteria were washed with 10mM NaCl and resuspended to obtain a Bacillus pumilus G15 resuspension (referred to as G15-resuspension 2).

[0087] Select potato tubers (fertile soil No. 5) of uniform size that are highly susceptible to aerial stem rot, wash them, disinfect them with sodium hypochlorite for 20 minutes, rinse them with sterile water, and dry them. Use a sterilized yellow gun tip to pierce the potato tubers to create cavities, then add 50 μL of FN20111-resuspended solution 1 and 50 μL of G15-resuspended solution 2 into the cavities, respectively, and set up 5 parallel experiments.

[0088] Buffer (10 mM NaCl) was added to the potato cavity as a negative control.

[0089] Afterwards, each group sealed the wound with vaseline and then wrapped the potato tubers with paper towels, spread them flat in a fresh-keeping box and sprayed them with sterile water with a sprayer to keep them moist. After sealing and culturing in the dark at room temperature for 48 to 72 hours, the toxicity of Pectobacillus and Bacillus was evaluated based on the weight of the immersed tissue. Figure 5The toxicity of diverse pectin bacteria and Bacillus pumilus on potato tubers, where A is the longitudinal section of tubers inoculated with diverse pectin bacteria FN20111 and Bacillus pumilus G15; B is the analysis of the difference in internal rot weight of tubers with different treatments.

[0090] The results showed that the various Pectinobacterium FN20111 caused potato tubers to rot to varying degrees, while Bacillus pumilus G15 did not cause rot to potato tubers ( Figure 5 The weight of the rotten part caused by Bacillus multiforme FN20111 was 3.62±0.26g ( Figure 5 B). The weight of the decayed part caused by Bacillus pumilus G15 was 0.17±0.03g ( Figure 5 There is no significant difference between the two, indicating that Bacillus pumilus G15 will not cause rot or other symptoms on potato tubers, and the antibacterial test on pathogens can be carried out.

[0091] (2) Determination of the effectiveness of Bacillus tuber control against potato aerial stem rot pathogens

[0092] In order to clarify whether the biocontrol Bacillus has a preventive or therapeutic effect on Pectobacterium on potato tubers, two groups of treatments were set up to determine the control effect of Bacillus on potato aerial stem rot on tubers. Specifically:

[0093] Treatment 1: Select potato tubers (fertile soil No. 5) of uniform size that are highly susceptible to aerial stem rot, wash them, disinfect them with sodium hypochlorite for 20 minutes, rinse them with sterile water, and dry them. Use a sterilized yellow gun tip to pierce the potato tubers to create a cavity, add 50μL FN20111-resuspended solution 1 to the cavity, culture them in the dark at 28℃ for 12 hours, and then inoculate 50μL G15-resuspended solution 2. Set up 5 parallel experiments.

[0094] Treatment 2: Select potato tubers (fertile soil No. 5) of uniform size that are highly susceptible to aerial stem rot, wash them, disinfect them with sodium hypochlorite for 20 minutes, rinse them with sterile water, and dry them. Use a sterilized yellow gun tip to pierce the potato tubers to create a cavity, add 50 μL of G15-resuspended solution 2 to the cavity, culture them in the dark at 28°C for 12 hours, and then inoculate 50 μL of FN20111-resuspended solution 1. Set up 5 parallel experiments.

[0095] Results Figure 6 It can be seen that the weight of the rotten part of the potato tuber caused by inoculating Bacillus pumilus G15 for 12 hours after inoculating Bacillus pumilus FN20111 is 3.18±0.29g. The weight of the rotten part of the potato tuber caused by inoculating Bacillus pumilus G15 for 12 hours after inoculating Bacillus pumilus FN20111 is 0.23±0.04g ( Figure 6In A), the rotten area was significantly reduced compared with the treatment of inoculating with diverse pectinobacterium FN20111 for 12 hours and then inoculating with G15 ( Figure 6 B), therefore, Bacillus pumilus G15 has an inhibitory effect on Pectinobacterium diversicolor FN20111.

[0096] 5. Potted plant protection effectiveness determination

[0097] In order to clarify the control effect of Bacillus pumilus G15 on potato potted plants against Pectinobacterium variegatum FN20111, it was selected to carry out control effect determination on potato potted plants. The test method is as follows:

[0098] (1) Preparation of resuspension: Activate Bacillus pumilus G15 on LB plate, culture in a 37°C constant temperature incubator for 24 hours, pick a single colony and place it in LB liquid culture medium, culture at 37°C, 200 rpm for 12-14 hours to prepare seed solution. Add the seed solution to liquid LB at a volume ratio of 1:100, and shake and culture until the OD value of the culture solution reaches 600 =1.0, the culture solution was centrifuged at 8500 rpm for 1 min to collect the cells, washed with physiological saline and resuspended to 10 7 CFU / mL, and obtain G15 resuspension.

[0099] The multi-pectin bacteria FN20111 was activated and streaked on the LB plate, cultured in a constant temperature incubator at 28°C for 24 hours, and then a single colony was picked and placed in LB liquid culture medium, and cultured at 28°C and 200 rpm for 12 to 14 hours to prepare seed solution. The seed solution was added to the liquid LB at a volume ratio of 1:100, and the culture was shaken and cultured until the OD of the culture solution reached 0. 600 =0.8, the culture solution was centrifuged at 8500 rpm for 1 min to collect the cells, washed with physiological saline and resuspended to 10 7 CFU / mL, and obtain FN20111 resuspension.

[0100] (2) Treatment 1: The potato variety selected was Wo Tu No. 5, which is highly susceptible to aerial stem rot. The tubers were cut into pieces and wrapped with talcum powder and planted in pots. Watering was performed every 7 days. After about 30 days of cultivation, 30 mL of G15 resuspension was sprayed on the stems. Seven days after spraying, diverse pectinobacterium FN20111 was inoculated.

[0101] The inoculation of FN20111 resuspension was performed by injection. The specific operation was as follows: the inoculation site was disinfected with 75% ethanol before injection, 30 μL of FN20111 resuspension was sucked into the base of the potato stem with a 1 mL syringe, and the injection wound was sealed with vaseline after the injection to prevent the wound from being contaminated.

[0102] Treatment 2 (negative control): Same as treatment 1, the only difference is that both the G15 resuspension and the FN20111 resuspension were replaced with normal saline.

[0103] Treatment 3 (positive control): Same as treatment 1, except that the FN20111 resuspension was inoculated and the Bacillus pumilus G15 resuspension was not sprayed.

[0104] Treatment 4 (drug control): Same as treatment 1, except that the resuspension of Pectobacillus microti FN20111 was inoculated, and the spraying of the resuspension of G15 was replaced by spraying of 4% kasugamycin.

[0105] Ten stems were inoculated in each treatment. The control effect of Bacillus was determined by comparing the length of soft rot in the longitudinal section of potato plant stems.

[0106] The antibacterial effect of Bacillus pumilus G15 on Pectinobacterium multiflorum FN20111 in potato potted plants is shown in Figure 7 It can be seen that the average internal rot length of treatment 1, which was sprayed with Bacillus brevis G15 and then inoculated with diverse pectin Bacillus FN20111, was 4.76±0.62cm. The average internal rot length of treatment 3, which was only inoculated with diverse pectin Bacillus FN20111 (positive control), was 10.1±0.51cm ( Figure 7 In middle B), the average protective effect of Bacillus pumilus G15 on Pectinobacterium diversifolium FN20111 was 52.87%. The average protective effect of 4% kasugamycin in treatment 4 on Pectinobacterium diversifolium FN20111 was 71.90%.

[0107] In summary, the present invention provides a biocontrol Bacillus strain Bacillus pumilus G15 for inhibiting potato aerial stem rot pathogens, and the Bacillus pumilus G15 of the present invention has a significant inhibitory effect on diverse pectin Bacillus FN20111. The present invention also provides a bacterial agent, and the results of the examples show that when the resuspension of Bacillus pumilus G15 is sprayed on a potato pot plant, the control effect of Bacillus pumilus G15 on diverse pectin Bacillus FN20111 is 52.87%.

[0108] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus pumilus G15, with a deposit number of CGMCC No.32839.

2. A bacterial agent, characterized in that It comprises the Bacillus pumilus G15 described in claim 1.

3. The bacterial agent according to claim 2, characterized in that: The effective viable count of Bacillus pumilus G15 in the bacterial agent is ≥1×10 7 CFU / mL.

4. The bacterial agent according to claim 2, characterized in that: The application form of the Bacillus pumilus G15 includes bacterial suspension and / or fermentation broth.

5. The bacterial agent according to claim 4, characterized in that: The preparation of the fermentation liquid comprises: inoculating the Bacillus pumilus G15 into a culture medium for culturing to obtain a fermentation liquid; The preparation of the bacterial suspension includes: centrifuging and resuspending the fermentation liquid to obtain the bacterial suspension.

6. Use of the Bacillus pumilus G15 according to claim 1 or the bacterial agent according to any one of claims 2 to 5 in controlling pathogenic bacteria of the genus Pectobacterium spp.

7. Use of Bacillus pumilus G15 according to claim 1 or the bacterial agent according to any one of claims 2 to 5 in preventing and controlling potato diseases.

8. The use according to claim 7, characterized in that: The diseases include potato blackleg and / or potato aerial stem rot.

9. A method for preventing and controlling potato diseases, characterized in that: The Bacillus pumilus G15 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 is applied to potatoes.

10. The method according to claim 9, characterized in that When used in the field, the application rate of the bacterial agent is 1.9-2.1L / 667m 2 When used for potted plants, the amount of the bacterial agent sprayed is 28 to 32 mL per seedling.

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