Preparation method and application of composite microbial agent for preventing and controlling rapeseed black shank pathogen

Abstract

The invention discloses a preparation method and application of a composite microbial agent for preventing and controlling rapeseed black shank pathogen, and belongs to the field of microbial agents. The biological pesticide of the invention is prepared by mixing Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis with inorganic salt ions in a certain proportion, can effectively inhibit the pathogenic bacteria of rapeseed black shank pathogen, and can provide technical support for the development of a new agent for preventing and controlling rapeseed black shank pathogen.

Description

Technical Field

[0001] The invention belongs to the field of microbial agents, and particularly relates to a preparation method and application of a composite microbial agent for preventing and controlling rapeseed black shank pathogen. Background Art

[0002] As a serious plant disease, rapeseed black shank poses a major threat to rapeseed production. The damage caused by rapeseed black shank is extensive and deep, affecting not only the roots, stems, leaves and siliques of rapeseed, but also other cruciferous vegetables such as cabbage and radish. Black shank can occur throughout the entire growth period of rapeseed, and is most serious in the later stages.

[0003] The harm of rapeseed black shank disease lies not only in the direct death of plants and yield reduction, but also in its long-term contamination of soil and seeds. The pathogen overwinters in seeds, soil or diseased residues as mycelium and conidia, becoming the primary source of infection the following year. Therefore, once black shank disease occurs in the field, long-term soil and seed treatment is required to effectively control the occurrence of the disease.

[0004] At present, the prevention and control of rapeseed black shank pathogen mainly relies on chemical control methods. Although the selection of varieties with strong disease resistance is one of the effective prevention and control measures, due to the rapid mutation rate of rapeseed black shank pathogen, the effectiveness of disease-resistant varieties is difficult to maintain in the long term, so it is necessary to continuously update and introduce new disease-resistant varieties. This method is not only time-consuming, but also has unstable effects. At the same time, the use of chemical agents may also bring about problems such as agent residues and environmental pollution, posing a potential threat to the ecological environment and human health. Therefore, biological control methods are gradually gaining attention as an environmentally friendly and sustainable alternative. In the future, biological control is expected to play a greater role in the prevention and control of rapeseed black shank pathogen, providing a more sustainable and environmentally friendly solution for the safe production of rapeseed.

[0005] Through searching the existing patent literature, it was found that CN201811435140.6 discloses a biocontrol fungus Simplicillium lamellicola P2. Although the filtrate of the strain after 15 days of cultivation can inhibit rapeseed black shank pathogens by more than 90%, the cultivation time is too long and it is not suitable for enterprise production. The solution proposed in the present invention can achieve the same effect after 2 days of cultivation. In addition, CN110184194A discloses a biocontrol bacterium Simplicillium lamellicola JC-1, but the inhibition rate of the strain on rapeseed black shank pathogens after 15 days of cultivation is only 44%, which cannot meet the application requirements. CN101385468A discloses a licheniformis strain that inhibits tobacco black shank pathogens, but it requires the number of live bacteria in the bacterial solution to reach 1.5*10 10 , which has higher requirements for high-density fermentation technology. Summary of the invention

[0006] In view of the above-mentioned prior art, the present invention provides a microbial agent for preventing and controlling rapeseed black shank pathogen and a preparation method thereof, and the produced microbial agent has a good prevention and control effect on rapeseed black shank pathogen, which is of great significance for preventing and controlling rapeseed black shank pathogen.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The invention relates to a liquid microbial composite bacterial agent, which contains a mixed bacterial liquid prepared from Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis.

[0009] As an embodiment, the volume ratio of the Bacillus subtilis suspension, the Bacillus amyloliquefaciens suspension and the Bacillus licheniformis suspension in the mixed bacterial liquid is 1:0.5-2:0.5-2.

[0010] As an embodiment, the total effective viable count in the mixed bacterial solution is 0.5-1*10 9 CFU / mL.

[0011] As an embodiment, the mixed bacterial liquid further contains at least one of sodium molybdate, copper sulfate, zinc sulfate, ferrous sulfate, magnesium sulfate, and potassium sulfate.

[0012] As an embodiment, the mixed bacterial liquid further contains sodium molybdate, copper sulfate, zinc sulfate, ferrous sulfate, magnesium sulfate and potassium sulfate.

[0013] As an embodiment, the mixed bacterial liquid contains 0.2-0.6 g / L sodium molybdate, 0.3-0.8 g / L copper sulfate, 0.1-0.5 g / L zinc sulfate, 0.2-0.6 g / L ferrous sulfate, 0.1-0.5 g / L magnesium sulfate and 0.2-0.6 g / L potassium sulfate.

[0014] The present invention also relates to a method for preparing a liquid microbial composite agent, the method comprising the following steps:

[0015] S1. Streak Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis respectively (in LB solid medium) and culture at a constant temperature;

[0016] S2, picking a single colony (inoculated in LB medium) and continuing to culture; obtaining Bacillus subtilis suspension, Bacillus amyloliquefaciens suspension and Bacillus licheniformis suspension respectively;

[0017] S3, mixing the Bacillus subtilis suspension, the Bacillus amyloliquefaciens suspension and the Bacillus licheniformis suspension.

[0018] As an embodiment, in step S1, the constant temperature culture temperature is 35-38°C and the time is 16-32h.

[0019] As an embodiment, in step S2, the culture conditions are 35-38° C. and 150-250 rpm for 16-32 hours.

[0020] In some implementation examples, in step S2, the effective viable counts of the obtained Bacillus subtilis suspension, Bacillus amyloliquefaciens suspension, and Bacillus licheniformis suspension are 2*10 8 , 2*10 8 , 2*10 8 CFU / mL.

[0021] As an embodiment, after step S3, the method further includes adding at least one of sodium molybdate, copper sulfate, zinc sulfate, ferrous sulfate, magnesium sulfate and potassium sulfate to the mixed bacterial liquid.

[0022] The invention also relates to the use of a liquid microbial composite agent in preventing and controlling plant diseases.

[0023] As an embodiment, the plant disease includes Phytophthora spp.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The composite microbial agent of the present invention is prepared by mixing Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis in a specific ratio, which can effectively inhibit the pathogenic bacteria of rapeseed black shank pathogen, and can provide support for the development of new agents for preventing and controlling rapeseed black shank pathogen.

[0026] (2) After 2 days of culturing, the mixed bacterial liquid prepared from Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis in the composite microbial agent of the present invention can achieve an inhibition rate of more than 90% against rapeseed black shank pathogen.

[0027] (3) The total effective viable count of the composite microbial agent of the present invention is 0.5-1*10 9 CFU / mL can achieve a good inhibitory effect, which is much lower than the number of viable bacteria disclosed in CN101385468A and other publications. DETAILED DESCRIPTION

[0028] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0029] The strains and culture media involved in the following examples are as follows:

[0030] 1. Test strains

[0031] (1) The pathogen of rapeseed black shank disease is Leptosphaeria Maculans, with the deposit number being ATCC 46318.

[0032] (2) Bacillus subtilis, deposit number is ATCC No. 202152.

[0033] (3) Bacillus amyloliquefaciens, deposit number is CGMCC No.1.281.

[0034] (4) Bacillus licheniformis, deposit number is CGMCC No.1.10257.

[0035] 2. Culture medium

[0036] LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of deionized water. For solid medium, 18 g of agar powder was added.

[0037] PDA medium: 200 g potato, 20 g glucose, KH 2 PO 4 3g, MgSO 4 .7H 2 O 1.5g, distilled water 1000mL. For solid medium, add 18g agar powder.

[0038] Embodiment: Preparation of microbial agent for controlling rapeseed black shank pathogen

[0039] (1) Preparation of Bacillus subtilis suspension: Bacillus subtilis previously stored in a 4°C refrigerator was inoculated onto LB solid culture medium by streaking. Subsequently, these culture media were placed in a constant temperature environment of 37°C for 24 hours. After obvious single colonies were observed, these single colonies were carefully picked up, seeded in LB liquid culture medium, and then inoculated into fresh LB culture medium (initial pH value was 7) at a ratio of 1% (v / v). After that, the culture was continued at 37°C and a rotation speed of 180 rpm for 24 hours, and the amount of live bacteria in the culture medium was adjusted to 2*10 with distilled water. 8 CFU / mL was used to obtain Bacillus subtilis suspension.

[0040] (2) Preparation of Bacillus amyloliquefaciens suspension: The Bacillus amyloliquefaciens previously stored in a refrigerator at 4°C was inoculated onto LB solid culture medium by streaking. Subsequently, the culture medium was placed in a constant temperature environment of 37°C for 24 hours. After obvious single colonies were observed, these single colonies were carefully picked up, seed cultured in LB liquid culture medium, and then inoculated into fresh LB culture medium (initial pH value was 7) at a ratio of 1% (v / v). After that, the culture was continued at 37°C and a rotation speed of 180 rpm for 24 hours, and the amount of live bacteria in the culture medium was adjusted to 2*10 with distilled water. 8 CFU / mL was used to obtain the Bacillus amyloliquefaciens suspension.

[0041] (3) Preparation of Bacillus licheniformis suspension: The Bacillus licheniformis previously stored in a 4°C refrigerator was inoculated onto LB solid culture medium by streaking. Subsequently, these culture media were placed in a constant temperature environment of 37°C for 24 hours. After obvious single colonies were observed, these single colonies were carefully picked up, seed cultured in LB liquid culture medium, and then inoculated into fresh LB culture medium (initial pH value was 7) at a ratio of 1% (v / v). After that, the culture was continued at 37°C and a rotation speed of 180 rpm for 24 hours, and the amount of live bacteria in the culture medium was adjusted to 2*10 with distilled water. 8 CFU / mL was used to obtain the Bacillus licheniformis suspension.

[0042] (4) Preparation of mixed bacterial solution: the volume ratio of Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis was 1:1:1 (Group A), 1:2:1 (Group B), 1:1:2 (Group C), and 2:1:1 (Group D).

[0043] Verification Example, Microbial Agent Inhibitory Effect on Pathogen of Rapeseed Black Leg Pathogen

[0044] (1) Relative inhibition rates of single bacterial suspensions of Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis against rapeseed black shank pathogen.

[0045] The rapeseed black leg pathogen was inoculated on PDA solid culture medium and cultured at 28°C for 60h. A bacterial cake was punched out from the edge of the colony with a sterile cork borer (diameter = 6mm) and inoculated in the center of a new PDA culture medium plate. 3 μL of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis suspension and two-by-two combinations (volume ratio 1:1) were inoculated on both sides 2 cm away from the bacterial cake. Sterile water was used as the control group. Each treatment was repeated three times. When the mycelium in the control group PDA culture medium plate covered the entire plate, the growth radius of the pathogen colony was measured, and the inhibition rate was calculated using the following formula.

[0046] Relative inhibition rate = (control colony radius - treated colony radius) / control colony radius × 100%.

[0047] (2) Relative inhibition rate of mixed bacterial suspension against rapeseed black leg pathogen

[0048] The relative inhibition rate of the mixed solution groups A, B, C and D was determined by the same operation as above.

[0049] (3) Relative inhibition rate of microbial agents on rapeseed black leg pathogen

[0050] The relative inhibition rate of the microbial agent was determined by the same operation as above. Preparation of microbial agent: 0.4 g / L sodium molybdate, 0.5 g / L copper sulfate, 0.3 g / L zinc sulfate, 0.4 g / L ferrous sulfate, 0.3 g / L magnesium sulfate and 0.5 g / L potassium sulfate were added to the mixed bacterial solution of group A.

[0051] (4) Relative inhibition rate of different dilution multiples of microbial agents on rapeseed black leg pathogen

[0052] The microbial agent in (3) was diluted 0 times, 5 times, 10 times, 50 times, 100 times, 200 times and 500 times with sterile water, and the relative inhibition rate of the solutions with different dilution times against rapeseed black leg pathogen was detected by the same method as above, and each treatment was repeated three times.

[0053] 5. Measurement results

[0054] (1) Relative inhibition rate of Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis suspensions in pairs against rapeseed black leg pathogen

[0055] Table 1 Antibacterial effect of the single bacterial suspension prepared by the present invention on rapeseed black leg pathogen

[0056] Substance to be tested Relative antibacterial rate (%) Bacillus subtilis suspension 59.6％ Bacillus amyloliquefaciens suspension 62.5％ Bacillus licheniformis suspension 56.8％ Bacillus subtilis + Bacillus amyloliquefaciens bacterial suspension 62.3％ Bacillus subtilis + Bacillus licheniformis suspension 58.9％ Bacillus amyloliquefaciens + Bacillus licheniformis suspension 63.2％

[0057] According to the data in Table 1, it can be clearly seen that the single bacterial suspension prepared by the present invention has a certain inhibitory effect on rapeseed black leg pathogen, but it is not very efficient. Among the two combinations, the combination of Bacillus amyloliquefaciens + Bacillus licheniformis bacterial suspension has the best effect, and the inhibition rate of rapeseed black leg pathogen can reach 63.2%.

[0058] (2) Relative inhibition rate of the mixed solution against rapeseed black leg pathogen

[0059] Table 2 Antibacterial effect of the mixed bacterial suspension prepared by the present invention on rapeseed black leg pathogen

[0060] Substance to be tested Relative antibacterial rate (%) Group A(1:1:1) 95.8％ Group B (1:2:1) 86.8％ Group C (1:1:2) 84.3％ Group D (2:1:1) 78.5％

[0061] According to the data in Table 2, it can be clearly seen that the mixed bacterial suspension prepared by the present invention has a good inhibitory effect on rapeseed black leg pathogen, among which group A (1:1:1) is as high as 95.8%, and the inhibitory effect on pathogenic bacteria is obvious.

[0062] (3) Relative inhibition rate of microbial agents against rapeseed black leg pathogen

[0063] Table 3 Relative inhibition rate of the microbial agent prepared by the present invention on rapeseed black leg pathogen

[0064] Substance to be tested Relative antibacterial rate (%) Microbial Agents 96.6％

[0065] According to the data in Table 3, it can be clearly seen that the microbial agent prepared by the present invention has a significant inhibitory effect on rapeseed black shank pathogen. After the addition of ions, its antibacterial activity is not affected, and there is a slight promoting effect. The antibacterial rate against rapeseed black shank pathogen can reach 96.6%.

[0066] (4) Relative inhibition rate of microbial agent dilution on rapeseed black leg pathogen

[0067] Table 4 Relative inhibition rate of the microbial agent dilution prepared by the present invention on rapeseed black leg pathogen

[0068] Mixtures with different dilution ratios Relative antibacterial rate (%) Original solution 96.6％ Dilute 5 times solution 95.8％ Dilute 10 times solution 94.6％ Dilute 50 times solution 91.5％ Dilute 100 times solution 88.8％ Dilute 200 times solution 75.4％ Dilute 500 times solution 68.2％

[0069] According to the data in Table 4, it can be seen that the 5-fold dilution, 10-fold dilution, 50-fold dilution, 100-fold dilution and 200-fold dilution of the mixture prepared by the present invention all have an antibacterial effect on rapeseed black leg pathogen. Although the antibacterial rate decreases with the increase of the dilution multiple, the antibacterial rate is still above 70%. This result shows that the microbial agent prepared by the present invention has a strong antibacterial ability and can effectively inhibit rapeseed black leg pathogen. Therefore, this mixture has broad application prospects in preventing and controlling rapeseed black leg pathogen caused by rapeseed black leg pathogen. Compared with the existing traditional chemical agents, the microbial agent provided by the present invention is non-toxic, non-polluting and residue-free, which ensures the safety of food; at the same time, it also effectively avoids the enhancement of rapeseed black leg pathogen resistance caused by long-term use of chemical agents.

[0070] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A liquid microbial composite agent, characterized in that: The bacterial agent contains a mixed bacterial solution, which is prepared from Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis.

2. The liquid microbial composite agent according to claim 1, characterized in that: The volume ratio of the Bacillus subtilis suspension, the Bacillus amyloliquefaciens suspension and the Bacillus licheniformis suspension in the mixed bacterial liquid is 1:0.5-2:0.5-2.

3. The liquid microbial composite agent according to claim 1, characterized in that: The mixed bacterial liquid also contains at least one of sodium molybdate, copper sulfate, zinc sulfate, ferrous sulfate, magnesium sulfate and potassium sulfate.

4. The liquid microbial composite agent according to claim 3, characterized in that: The mixed bacterial liquid contains 0.2-0.6 g / L sodium molybdate, 0.3-0.8 g / L copper sulfate, 0.1-0.5 g / L zinc sulfate, 0.2-0.6 g / L ferrous sulfate, 0.1-0.5 g / L magnesium sulfate and 0.2-0.6 g / L potassium sulfate.

5. A method for preparing the liquid microbial composite agent according to claim 1, characterized in that: The method comprises the following steps: S1, streak and culture Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis at a constant temperature respectively; S2, picking a single colony and continuing to culture, to obtain Bacillus subtilis suspension, Bacillus amyloliquefaciens suspension and Bacillus licheniformis suspension respectively; S3, mixing the Bacillus subtilis suspension, the Bacillus amyloliquefaciens suspension and the Bacillus licheniformis suspension.

6. The method for preparing the liquid microbial composite agent according to claim 5, characterized in that: In step S1, the constant temperature culture is at 35-38°C and the time is 16-32 hours.

7. The method for preparing the liquid microbial composite agent according to claim 5, characterized in that: In step S2, the culture conditions are 35-38° C. and 150-250 rpm for 16-32 hours.

8. The method for preparing the liquid microbial composite agent according to claim 5, characterized in that: After step S3, the method further includes adding at least one of sodium molybdate, copper sulfate, zinc sulfate, ferrous sulfate, magnesium sulfate and potassium sulfate to the mixed bacterial liquid.

9. Use of the liquid microbial composite agent according to claim 1 in preventing and controlling plant diseases.

10. The use according to claim 9, characterized in that The plant diseases include blackleg pathogen.