Application of mutant strain of bacillus subtilis in killing oncomelania and mutation method

Abstract

The invention relates to the technical field of parasite prevention and control, in particular to application of a mutant strain of bacillus subtilis in oncomelania killing and a mutagenesis method. According to the method, a mutant strain of bacillus subtilis is used for killing oncomelania; the mutant strain is any one of ARTP-129 or ARTP-154 or a combination of ARTP-129 and ARTP-154; the ARTP-129 is preserved in the China Center for Type Culture Collection on January 6, 2025, the preservation number of the ARTP-129 is CCTCC M 2025036, and the ARTP-154 is preserved in the China Center for Type Culture Collection on January 6, 2025, the preservation number of the ARTP-154 is CCTCC M 2025037. According to the technical scheme, the mutant strain of the bacillus subtilis is used for killing oncomelania, and the method is beneficial to improving the oncomelania killing efficiency and has the advantages of being environmentally friendly, free of harmful residues and capable of improving the environment.

Description

Technical Field

[0001] The present invention relates to the technical field of parasite prevention and control, specifically to the application of a mutagenized strain of Bacillus subtilis in killing Oncomelania hupensis and a mutagenesis method. Background Art

[0003] Since the 1950s, China has started the research on Oncomelania hupensis-killing microorganisms. Currently, dozens of Oncomelania hupensis-killing microorganisms have been discovered, such as Pseudomonas convexa, Bacillus cereus B59, Streptomyces violaceoruber, and Xanthobacter autotrophicus. Some Oncomelania hupensis-killing microorganisms have been studied thoroughly. However, due to the relatively strict application conditions of most microbial Oncomelania hupensis-killing agents or the difficulty in in vitro synthesis and large-scale production in the prior art, there are still great difficulties in field application, and the Oncomelania hupensis-killing efficiency is low.

[0004] Atmospheric and Room Temperature Plasma (ARTP) mutagenesis technology is a physical mutagenesis technology. Its mutagenesis principle is to use active energy particles to damage the genetic material of the strain, thereby inducing the strain cells to initiate the SOS mechanism, and finally obtaining a genetically stable mutagenized strain. Compared with traditional physical mutagenesis and chemical mutagenesis, ARTP mutagenesis technology has the advantages of higher mutagenesis performance, wider application range, more diverse damage mechanisms to genetic material, safe operation, and mild environment. Currently, it has been successfully applied to the mutagenesis breeding of various microorganisms such as bacteria, fungi, and microalgae. The successful application of ARTP mutagenesis technology in microbial breeding can be invested in the research of transforming Oncomelania hupensis-killing microorganisms to improve their Oncomelania hupensis-killing effect, introducing this technology, and thus developing a new type of Oncomelania hupensis-killing agent. Summary of the Invention

[0005] The present invention provides the application of a mutagenized strain of Bacillus subtilis in killing Oncomelania hupensis and a mutagenesis method to solve the problems in the related technologies. Using the mutagenized strain of Bacillus subtilis to kill Oncomelania hupensis is beneficial to improving the Oncomelania hupensis-killing efficiency, and has the advantages of being green and environmentally friendly, having no harmful residues, and improving the environment.

[0006] To solve the above problems, the following technical solutions are provided:

[0007] The application of the mutagenized strain of Bacillus subtilis of the present invention in killing Oncomelania hupensis uses the mutagenized strain of Bacillus subtilis to kill Oncomelania hupensis; the mutagenized strain is any one or a combination of ARTP-129 or ARTP-154.

[0008] Through the above solution, a mutagenized strain of Bacillus subtilis is used to kill Oncomelania hupensis. The Bacillus subtilis is mutagenized to obtain a mutagenized strain, and this mutagenized strain is used to kill Oncomelania hupensis. Bacillus subtilis is a commonly used biocontrol bacterium, which has the advantages of being green and environmentally friendly, having no harmful residues, and improving the environment. In this invention, Bacillus subtilis 3-4 is used as the experimental object, and it is mutagenized by ARTP to obtain mutagenized strains ARTP-129 and ARTP-154 with high Oncomelania hupensis killing effects, thus greatly improving the efficiency of Oncomelania hupensis control.

[0009] The ARTP-129 was deposited at the China Center for Type Culture Collection on January 6, 2025, with the deposit number CCTCC M 2025036 and the taxonomic name Bacillus subtilis ARTP-129.

[0010] The ARTP-154 was deposited at the China Center for Type Culture Collection on January 6, 2025, with the deposit number CCTCC M 2025037 and the taxonomic name Bacillus subtilis ARTP-154.

[0011] The ARTP-129 solution is used to immerse and kill Oncomelania hupensis, and the ARTP-129 solution is prepared by diluting the ARTP-129 fermentation broth with water.

[0012] In the above solution, when the ARTP-129 solution is used to immerse and kill Oncomelania hupensis, the mortality rate of Oncomelania hupensis reaches 85%.

[0013] The ARTP-154 solution is used to immerse and kill Oncomelania hupensis, and the ARTP-154 solution is prepared by diluting the ARTP-154 fermentation broth with water.

[0014] In the above solution, when the ARTP-154 solution is used to immerse and kill Oncomelania hupensis, the mortality rate of Oncomelania hupensis reaches 85%.

[0015] The mutagenesis method of the mutagenized strain of Bacillus subtilis includes the following steps:

[0016] S1: Absorb the activated Bacillus subtilis bacterial solution and inoculate it into a shake flask of LB liquid medium at an inoculation amount of 1%, and place it in a constant temperature shaker for shaking culture; samples are taken every 2 hours within 32 hours, and samples are taken every 4 hours after 32 hours;

[0017] S2: Centrifuge the bacterial solution in S1 to obtain a bacterial solution precipitate, wash the bacterial solution precipitate with sterile physiological saline, and then dilute the concentration of the bacterial solution precipitate with sterile physiological saline. The diluted bacterial solution precipitate is used as a bacterial suspension;

[0018] S3: Sterilize the metal slide, aspirate the bacterial suspension and evenly coat it on the surface of the sterile slide. Place the metal slide in an atmospheric room temperature plasma mutagenesis instrument to perform mutagenesis treatment on the bacterial suspension to obtain a mutagenized bacterial suspension;

[0019] S4: Dilute the mutagenized bacterial suspension, take the diluted mutagenized bacterial suspension and evenly coat it on the solid medium, and place it in a constant temperature incubator for cultivation. According to the growth situation of the strains, obtain a mutagenesis lethality curve;

[0020] S5: Select the optimal mutagenesis treatment time according to the mutagenesis lethality curve to obtain mutagenized strains.

[0021] The temperature of the constant temperature shaker in S1 is 30 °C and the rotation speed is 200 rpm / min.

[0022] The centrifugation time in S2 is 5 min, the rotation speed is 5000 rpm / min, and the dilution concentration of the bacterial liquid is 10 6 ~10 8 CFU / mL.

[0023] The sterilization treatment method in S3 is to burn the metal slide on the flame for 10 s and then cool it; the incident power of the mutagenesis treatment is 100 W, the gas flow rate is 10 SLM, the irradiation distance is 2 mm, and the treatment time is 20 - 25 s.

[0024] In S4, the mutagenized bacterial suspension is diluted 10 -3 ~10 -6 times; the cultivation temperature in the constant temperature incubator is 30 °C and the cultivation time is 24 h.

[0025] Adopting the above scheme, there are the following specific advantages:

[0026] The mutagenized strains of Bacillus subtilis in the present invention kill Oncomelania hupensis by using the method of biological snail control. Compared with the physical snail control method and the chemical snail control method, the biological snail control has less investment, smaller project volume, saves time and effort, has a fast snail control effect, a relatively wide application range, and the mutagenized strains of Bacillus subtilis 3 - 4 applied in the invention have the advantages of being green, non-toxic, highly efficient and stable. The mutagenized strains of Bacillus subtilis can kill Oncomelania hupensis, thereby controlling the spread of schistosomiasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to the specific embodiments of the present invention in combination with the drawings, wherein:

[0028] Figure 1 is the growth curve of Bacillus subtilis 3 - 4;

[0029] Figure 2 is the lethality curve graph of ARTP mutagenesis on Bacillus subtilis 3 - 4;

[0030] Figure 3 It is the effect diagram of the snail-killing of the fermentation broth of the mutagenized strain in LB for 24 h. Specific implementation manners

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all 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.

[0032] In specific embodiment 1, Bacillus subtilis 3-4 is selected as the raw material. Bacillus subtilis 3-4 was deposited at the China Center for Type Culture Collection on January 6, 2025, with the deposit number CCTCC M2025035 and the taxonomic name Bacillus subtilis 3-4.

[0033] The first step: Select materials and preparation work

[0034] The experimental strain is Bacillus subtilis 3-4, and the experimental object is Oncomelania hupensis. After the snails captured in the wild are transported to the laboratory, they are rinsed three times with dechlorinated water, and then placed in a tray lined with a moist sponge pad and grass paper, and raised at room temperature (26±1°C) for 7-10 days to adapt to the environment. Select adult snails with 6-8 whorls, non-infected and with better vitality for the experiment. Before the experiment, the experimental snails are washed 3 times with ultrapure water and reserved;

[0035] Experimental reagents: peptone, yeast extract, sodium chloride and agar powder;

[0036] Experimental instruments: constant temperature incubator, constant temperature shaking incubator, multifunctional microplate reader and atmospheric room temperature plasma mutagenesis instrument.

[0037] The second step: Prepare ARTP-129 and ARTP-154

[0038] 2.1. Plot the growth curve of Bacillus subtilis 3-4:

[0039] Absorb the activated bacterial liquid and inoculate it into a shake flask of LB liquid medium at an inoculation amount of 1% (V / V). The liquid loading amount is 100 mL / 250 mL LB, and it is oscillated and cultured in a constant temperature shaker at 30°C and 200 rpm / min; Samples are taken every 2 h, and every 4 h after 32 h. The LB liquid medium without inoculated bacteria is used as a control. The OD of the fermentation broth is measured using a microplate reader at a wavelength of 600 nm 600 value, record the sampling time and OD 600 value, repeat three times, with time (h) as the abscissa and OD600 The growth curve of Bacillus subtilis 3-4 was plotted with the value as the ordinate.

[0040] As Figure 1 shown, the growth curve of Bacillus subtilis 3-4 was measured, and the growth of Bacillus subtilis 3-4 could be divided into different periods; the growth curve of Bacillus subtilis 3-4 showed that it was in the lag phase in the first 6 h, entered the logarithmic phase after 6 h, and the cell concentration reached the maximum at the 44th h; the cell viability in the logarithmic phase was relatively high, which was more conducive to screening positive mutagenesis strains. Therefore, the bacterial suspension prepared from the bacterial liquid cultured for 30 h was selected for mutagenesis treatment.

[0041] 2.2. Preparation of bacterial suspension:

[0042] According to the growth curve, the bacterial liquid in the middle and late logarithmic growth phase was taken, centrifuged at 5000 rpm / min for 5 min to obtain the bacterial liquid precipitate, washed the bacterial liquid precipitate twice with sterile normal saline, and then diluted the bacterial liquid precipitate with sterile normal saline. The dilution concentration was 10 6 ~10 8 CFU / mL as the bacterial suspension, and the concentration of the bacterial suspension was measured by the plate counting method.

[0043] 2.3. ARTP mutagenesis:

[0044] The metal slide was sterilized by burning on the flame for 10 s. After cooling, 10 μL of the bacterial suspension was aspirated and evenly spread on the surface of the sterile slide. The metal slide was placed in an atmospheric room temperature plasma mutagenesis instrument with an incident power of 100 W, a gas flow rate of 10 SLM, and an irradiation distance of 2 mm. The bacterial suspension was treated for 0 s, 10 s, 20 s, 30 s, 40 s, 50 s, and 60 s respectively. The mutagenized slide was placed in 990 μL of sterile normal saline and shaken thoroughly on a vortex oscillator to wash off the bacterial species to obtain the mutagenized bacterial suspension.

[0045] 2.4. Calculation of mutagenic lethality

[0046] The mutagenized bacterial suspension was diluted 10 -3 , 10 -4 , 10 -5 , 10 -6 times respectively. 100 μL of the mutagenized bacterial suspension with a suitable dilution factor was taken and evenly spread on the solid medium, and 3 parallels were set for each gradient. It was inverted and placed in a constant temperature incubator at 30 °C. After culturing for 24 h, colony counting was carried out by the colony counting method to obtain the number of colonies at different mutagenic times. The bacterial liquid without mutagenesis treatment was used as a control, and the mutagenic lethality was calculated according to formula (1) to determine the optimal mutagenic time. The mutagenic lethality curve was plotted to select the appropriate mutagenic time.

[0047]

[0048] 2.5. Evaluation of the snail-killing effect of mutagenized strains

[0049] 2.5.1 Preparation of the test sample solution

[0050] The activated mutagenized strain was inoculated into LB liquid medium and cultured in a constant temperature shaker at 30 °C and 200 rpm / min for 24 - 30 h to obtain LB fermentation broth, and the OD of the LB fermentation broth was adjusted 600 to 0.4 ± 0.1. The LB fermentation broth was respectively prepared into test solutions at 50.00 mL / L.

[0051] 2.5.2 Immersion killing method

[0052] The immersion killing method was used to evaluate the snail-killing effect of the mutagenized strain. The test sample solutions prepared in 2.5.1 were filled into different wells of a six-well plate (13 mL / well), and 20 test Oncomelania hupensis were placed in each well. At the same time, an ultrapure water control was set. After immersion for 72 h, the snails were taken out respectively, washed 3 times with ultrapure water, and then placed in a six-well plate lined with a moist sponge pad and tissue paper. After recovery and rearing at 26 °C for 72 h, the death situation of the snails was identified, and the experiment was repeated 3 times.

[0053] 2.5.3 Method for identifying the death of Oncomelania hupensis

[0054] The Oncomelania hupensis after the experiment in 2.5.2 was placed in ultrapure water, and it was observed whether there was a soft body protruding from the experimental Oncomelania hupensis. Those with a soft body protruding were live snails, and for those without a soft body protruding, the tapping method was used to judge life and death, which were divided into three categories: dead snails (no soft body protruding, and no contraction reaction after being crushed), dying snails (no soft body protruding, but there is a contraction reaction after being crushed), and live snails (soft body protruding, and there is a contraction reaction after being crushed). Record the number of dead snails in each group, and calculate the mortality rate of Oncomelania hupensis. The calculation formula (2) of the mortality rate of Oncomelania hupensis is as follows:

[0055]

[0056] The calculation of the improvement rate of the snail-killing effect of the mutagenized strain refers to formula (3):

[0057]

[0058] 2.6. Lethality curve of ARTP mutagenesis treatment on Bacillus subtilis 3 - 4

[0059] The lethality rate is an important reference basis in the mutagenesis experiment. After Bacillus subtilis 3 - 4 was treated with ARTP mutagenesis for different times, the mortality rates of Bacillus subtilis were different to varying degrees, such as Figure 2As shown, with the extension of the treatment time, the lethality of Bacillus subtilis 3-4 increased. When the mutagenesis time was 10 s, the average lethality was 20.12%, and when it was 20 s, the average lethality was as high as 95.38%. There was a positive correlation between the mutagenesis rate of the bacteria and the mutagenesis dose. When the lethality was above 90%, the positive mutagenesis rate was relatively high. However, if the mutagenesis time was too long, some excellent mutagenic strains might be killed, thus affecting the screening results. Therefore, 20 s was selected as the optimal mutagenesis treatment time, and 214 mutagenic strains were obtained.

[0060] Step 3: Experimental data

[0061] As Figure 3 shown in Table 1, the mortality of Oncomelania hupensis after 72 h of immersion treatment with the solution prepared by diluting the 24-h LB fermentation broth of the mutagenic strains and Bacillus subtilis 3-4 to 50 mL / L. After χ 2 test, the differences in the mortality of Oncomelania hupensis between all treatment groups and the ultrapure water control group were statistically significant. When the mortality of Oncomelania hupensis caused by the mutagenic strains was ≤ 33.33% or ≥ 70.00%, the differences in the average mortality between Bacillus subtilis 3-4 and the mutagenic strains were statistically significant (χ 2 = 4.126, P = 0.042; χ 2 = 4.232, P = 0.040). Among the 72 mutagenic strains with better snail-killing effects than Bacillus subtilis 3-4, there were 15 mutagenic strains with statistically significant differences in the mortality of Oncomelania hupensis compared with Bacillus subtilis 3-4. Among them, strains ARTP-129 and ARTP-154 had the best snail-killing effects, and the average mortality of Oncomelania hupensis reached 85.00%. Compared with Bacillus subtilis 3-4, the improvement rate of the snail-killing effect reached 64.51%.

[0062] Table 1 Snail-killing effects of the 24-h LB fermentation broth of the mutagenic strains

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] To sum up, in the experiment of killing Oncomelania hupensis by the immersion method, the mutagenic strains of Bacillus subtilis 3-4 were used to immerse Oncomelania hupensis. Among them, strains ARTP-129 and ARTP-154 had the best snail-killing effects. When using ARTP-129 to immerse Oncomelania hupensis, the mortality of Oncomelania hupensis reached 85%, and when using ARTP-154 to immerse Oncomelania hupensis, the mortality of Oncomelania hupensis reached 85%.

[0069] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, based on the above description, other different forms of changes or variations can be made. It is not necessary and impossible to list all the implementation manners here, and the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. The use of a mutagenic strain of Bacillus subtilis in killing Oncomelania snails is characterized in that: The mutagenic strain of Bacillus subtilis was used to kill Oncomelania snails.

2. The use of the mutated strain of Bacillus subtilis according to claim 1 in killing Oncomelania snails, characterized in that: The mutagenic strain is any one or a combination of ARTP-129 or ARTP-154; The ARTP-129 was deposited in the China Center for Type Microorganism Collection on January 6, 2025, with the deposit number of CCTCC M2025036; The ARTP-154 was deposited in the China Center for Type Microorganism Collection on January 6, 2025, with the deposit number CCTCC M2025037.

3. The use of the mutated strain of Bacillus subtilis in killing Oncomelania snails as claimed in claim 2, characterized in that: The snails are killed by immersing an ARTP-129 solution, wherein the ARTP-129 solution is prepared by diluting the ARTP-129 fermentation liquid with water.

4. The use of the mutated strain of Bacillus subtilis in killing Oncomelania snails as claimed in claim 2, characterized in that: The snails are killed by immersing an ARTP-154 solution, wherein the ARTP-154 solution is prepared by diluting the ARTP-154 fermentation liquid with water.

5. The mutagenesis method of the mutagenic strain of Bacillus subtilis according to claim 2, characterized in that: The following steps are involved: S1: The activated Bacillus subtilis liquid was inoculated into a LB liquid medium shake flask at a 1% inoculum amount, and the flask was placed in a constant temperature shaker for shaking culture; samples were taken every 2 hours within 32 hours, and samples were taken every 4 hours after 32 hours; S2: centrifuging the bacterial solution in S1 to obtain a bacterial solution precipitate, washing the bacterial solution precipitate with sterile saline, and then diluting the bacterial solution precipitate with sterile saline, and the diluted bacterial solution precipitate is used as a bacterial suspension; S3: sterilizing the metal slide, drawing the bacterial suspension and evenly coating it on the surface of the sterile slide, placing the metal slide in a normal pressure and room temperature plasma mutagenizer, performing mutagenesis on the bacterial suspension, and obtaining a mutagenic bacterial suspension; S4: diluting the mutagenic bacterial suspension, evenly coating the diluted mutagenic bacterial suspension on a solid culture medium, culturing in a constant temperature incubator, and obtaining a mutagenic lethality curve based on the growth of the strain; S5: According to the mutagenesis lethality curve, the optimal mutagenesis treatment time is selected to obtain the mutagenized strain.

6. The mutagenesis method of the mutagenic strain of Bacillus subtilis according to claim 5, characterized in that: The temperature of the constant temperature shaker in S1 was 30°C and the rotation speed was 200 rpm / min.

7. The method for mutagenizing a mutagenized strain of Bacillus subtilis according to claim 5, characterized in that: The centrifugation time in S2 was 5 min, the speed was 5000 rpm / min, and the concentration of the diluted bacterial solution was 10 6 ~10 8 CFU / mL.

8. The method for mutagenizing a mutagenized strain of Bacillus subtilis according to claim 5, wherein: The sterilization method in S3 is to burn the metal slide on the flame for 10 seconds and then cool it down; the incident power of the mutagenesis treatment is 100W, the gas flow rate is 10SLM, the irradiation distance is 2mm, and the treatment time is 20 to 25 seconds.

9. The method for mutagenizing a mutagenized strain of Bacillus subtilis according to claim 5, wherein: Dilute the mutagenic bacterial suspension 10 -3 ~10 -6 times; the culture temperature in the constant temperature incubator is 30℃ and the culture time is 24h.

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