Biopesticide mixture for preventing and treating wheat stripe rust and application thereof

Abstract

The invention discloses a biopesticide mixture for preventing and treating wheat stripe rust and application of the biopesticide mixture, and belongs to the technical field of agricultural prevention and treatment. The biopesticide is formed by mixing the bacillus subtilis suspension and the stellera chamaejasme root extract according to the dosage ratio of 100 mL: (2-10) g, pathogenic bacteria of wheat stripe rust can be effectively inhibited, the prevention and treatment effect of a mixed agent stock solution on wheat stripe rust can reach 94%, the prevention and treatment effect can still reach 81% after the mixed agent stock solution is diluted by 50 times, and the application range is wide. A support can be provided for developing a novel medicament for preventing and treating the wheat stripe rust.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural prevention and control, and specifically relates to a biological pesticide mixture for preventing and controlling wheat stripe rust and application thereof. Background Art

[0002] Wheat stripe rust is one of the most important wheat diseases in the world and an important biological disaster that affects the safe production of wheat in China. Wheat stripe rust can damage wheat leaves, weaken photosynthesis, and reduce nutrient absorption, thereby hindering wheat growth and development and reducing yields. In years when wheat stripe rust is prevalent, wheat production can be reduced by more than 40%, or even total crop failure. Wheat stripe rust can cause wheat grains to be incomplete and of reduced quality, affecting the selling price. In order to prevent and control wheat stripe rust, growers need to invest a lot of manpower, material resources, and financial resources, which increases planting costs.

[0003] At present, the prevention and control methods of wheat stripe rust mainly focus on chemical control. Selecting wheat varieties with strong disease resistance is one of the effective ways to prevent and control wheat stripe rust. However, due to the rapid mutation rate of stripe rust bacteria, disease-resistant varieties are often difficult to be effective continuously, so disease-resistant varieties need to be continuously updated. Therefore, this method takes a long time and is unstable. In addition, the use of chemical agents will lead to problems such as agent residues and environmental pollution. Therefore, biological control methods are a promising alternative.

[0004] After searching the existing patent documents, it was found that CN1934241 discloses a Bacillus subtilis EB120, which has high antagonistic activity for preventing and treating various plant diseases, and the plant diseases are selected from barley powdery mildew, cucumber powdery mildew, pepper anthracnose, rice blast, tomato gray mold, tomato late blight and wheat leaf rust. The strain can be used as a microbial agent for biological control of plant diseases. The ethyl acetate extract of the culture solution of Bacillus subtilis EB120 has a control effect of only 53% on wheat leaf rust, and the control effect of the culture supernatant can reach 90%. However, it directly uses the cultured stock solution, and it is impossible to know the effect on wheat leaf rust after dilution by a certain multiple, which does not meet the dilution requirements in practical applications, and it is even more impossible to guide the prediction of the control effect of wheat stripe rust.

[0005] The background technology of the CN1544433A specification states that in the "Chinese Native Pesticides" published in 1959, the water extract of wolfsbane root is used in the prevention and treatment of some plant diseases (including leaf rust). However, the main active ingredients of the root of wolfsbane are more soluble in the organic phase, and the water extract contains only a small part of the active ingredients. The content will be lower during the dilution process of the pesticide, and the use of the water extract cannot exert its maximum function. For this reason, CN1544433A also adopts a solvent extraction scheme. Further, CN1544433A proposes a method for extracting new wolfsbane toxin B from the root of wolfsbane, indicating that the new wolfsbane toxin B has an inhibition rate of more than 80% at a concentration of 2200 mg / L on plant pathogens such as apple dry rot fungi, wheat fusarium wilt fungi, tomato early blight fungi, pumpkin wilt fungi, corn leaf blight fungi, tobacco red star fungi, and pepper phytophthora fungi. However, the inhibitory effect on wheat stripe rust was not tested, and the concentration was too high (2200 mg / L), far exceeding the actual application concentration. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a biological pesticide mixture for controlling wheat stripe rust, which has practical application value and can meet the dilution requirements during use, and its application; the present invention prepares the mixture by mixing a Bacillus subtilis suspension and a root extract of Stellera chamaejasme, which can inhibit the growth of the wheat-specific type of Puccinia stripe fungus, and is of great significance for controlling wheat stripe rust.

[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 is prepared by mixing a bacillus subtilis suspension with a root extract of stellera chamaejasme.

[0009] As an embodiment of the present invention, the usage ratio of the Bacillus subtilis suspension to the root extract of Stellera chamaejasme is 10 mL: 1-12 g, preferably 10 mL: 2-12 g, and more preferably 10 mL: 2-10 g.

[0010] As an embodiment of the present invention, the Bacillus subtilis is Bacillus subtilis ATCC No.6633.

[0011] As an embodiment of the present invention, the number of viable bacteria in the Bacillus subtilis suspension is 1×10 8 -1×10 4 CFU / mL.

[0012] As an embodiment of the present invention, the root extract of Stellera chamaejasme is prepared by a method comprising the following steps:

[0013] A1. Dry and crush the roots of Stellera chamaejasme; mix the powder with 70-100wt.% ethanol, and soak them to form a preliminary ethanol soaking solution;

[0014] A2, filtering, separating the soaked material of the root of Stellera chamaejasme, and adding it to 70-100wt.% ethanol for soaking again; the obtained ethanol soaking liquid is mixed with the preliminary ethanol soaking liquid to obtain the target ethanol soaking liquid;

[0015] A3. Ultrasonic extraction: After filtering, an ethanol extract of the root of Stellera chamaejasme is obtained.

[0016] As an embodiment of the present invention, in step A1, the solid-liquid ratio of powder to ethanol is 1 g:10-30 mL, and the soaking time is 10-16 hours.

[0017] As an embodiment of the present invention, in step A2, the solid-liquid ratio of the root soaked material to 70-100 wt.% ethanol is 1 g: 3-5 mL.

[0018] As an embodiment of the present invention, in step A3, the ultrasonic extraction is performed 2-5 times at 25-50°C, the extraction parameters are 200-500W, 20KHZ, and the extraction time for each time is 20-40 minutes.

[0019] As an embodiment of the present invention, step A3 further comprises a purification step:

[0020] A4, filtering and concentrating the ethanol extract under reduced pressure to obtain an ethanol extract;

[0021] A5, adding the ethanol extract to an ethanol aqueous solution with a concentration of 20-40wt.% to dissolve it to obtain a pre-extraction solution;

[0022] A6. Extract continuously for multiple times using ethyl acetate at room temperature, and mix the obtained extracts; filter and concentrate under reduced pressure to obtain the target extract.

[0023] As an embodiment of the present invention, the Bacillus subtilis suspension is prepared by a method comprising the following steps:

[0024] B1. Streak Bacillus subtilis onto LB solid medium and culture at a constant temperature;

[0025] B2. Pick a single colony and inoculate it into LB medium, and culture it at 35-38°C and 150-250rpm for 16-32h to obtain seed solution;

[0026] B3. Transfer the seed solution to new LB medium at an inoculum rate of 1%, and culture at 35-38° C. and 150-250 rpm for 16-48 h to obtain a Bacillus subtilis suspension.

[0027] In some implementation examples, a single colony is picked and inoculated into LB medium, and cultured at 37°C and 180 rpm for 24 hours to obtain a seed solution; the seed solution is transferred to a new LB medium at an inoculation rate of 1%, and cultured at 37°C and 180 rpm for 48 hours to obtain a Bacillus subtilis suspension.

[0028] The invention also relates to the use of a liquid microbial composite agent in preventing and controlling plant diseases.

[0029] As an embodiment of the present invention, the plant disease includes wheat stripe rust.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1) The biopesticide of the present invention is prepared by mixing a Bacillus subtilis suspension with a root extract of Stellera chamaejasme in a dosage ratio of 100 mL: 2-10 g, which can effectively inhibit the pathogenic bacteria of wheat stripe rust and provide support for the development of new agents for preventing and controlling wheat stripe rust.

[0032] 2) The control effect of the mixed agent stock solution provided by the present invention on wheat stripe rust can reach 94%, and can still reach 81% after being diluted 50 times, which can meet the dilution requirements in practical applications. DETAILED DESCRIPTION

[0033] 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.

[0034] The strains and culture media involved in the following examples are as follows:

[0035] 1. Test strains

[0036] (1) The pathogen of wheat stripe rust (Puccinia striatum wheat-specific type) was collected from wheat leaves showing stripe rust at a high-quality wheat planting base in Zhangshi Town, Weishi County, Kaifeng City, Henan Province.

[0037] (2) Bacillus subtilis, deposited with ATCC No. 6633 and 168.

[0038] 2. Culture medium

[0039] 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.

[0040] PDA medium: 200 g potato, 20 g glucose, 3 g KH2PO4, 1.5 g MgSO4.7H2O, 1000 mL distilled water. For solid medium, add 15 g agar powder.

[0041] Embodiment, preparation of mixture for preventing and controlling wheat stripe rust

[0042] (1) Preparation of Bacillus subtilis suspension: Bacillus subtilis ATCC No. 6633 stored in a refrigerator at 4°C was streaked onto LB solid medium and cultured at 37°C for 24 hours. A single colony was then picked and inoculated into a fresh LB medium (with an initial pH value of 7), and cultured at 37°C and 180 rpm for 24 hours to obtain a seed solution. The seed solution was transferred to a new LB medium (with an initial pH value of 7) at an inoculation rate of 1%, and cultured at 37°C and 180 rpm for 48 hours to obtain a Bacillus subtilis suspension with a viable cell count of about 2*10 9 ;

[0043] (2) Preparation of root extract of Stellera chamaejasme: The root of Stellera chamaejasme was placed in an oven and dried at 35°C until its mass no longer changed. Then, the dried root was crushed and sieved through a 300-mesh standard sieve to obtain Stellera chamaejasme root powder. Then, the powder was mixed with 95% ethanol at a solid-liquid ratio of 1:10-30 (g:mL) and soaked for 10-16 hours to form a preliminary ethanol soaking solution. The preliminary ethanol soaking solution was filtered to separate the Stellera chamaejasme root soaking material and the soaked ethanol. The root soaking material was then added to 95% ethanol at a solid-liquid ratio of 1:3-5, and the obtained ethanol soaking solution was mixed twice to obtain the target ethanol soaking solution. Next, the target ethanol soaking solution was subjected to ultrasonic extraction 3 times at 40°C, each extraction time being 30 minutes. After filtration, the ethanol extract of Stellera chamaejasme root was obtained. For further purification, the ethanol extract was filtered and concentrated under reduced pressure using a rotary evaporator to obtain an ethanol extract. The ethanol extract was dissolved in a 30% ethanol aqueous solution to obtain a pre-extraction solution. Finally, the pre-extraction solution was extracted five times with ethyl acetate at room temperature, and the extracts obtained from the five extractions were mixed. The extract was filtered and concentrated under reduced pressure using a rotary evaporator to obtain the target extract.

[0044] (3) Preparation of a mixture: The Bacillus subtilis suspension and the root extract of Stellera chamaejasme are mixed in a dosage ratio of 100 mL: 2-10 g to obtain a mixture for preventing and controlling wheat fusarium head blight. The dosage ratio is 100 mL: 1 g for case 1; the dosage ratio is 100 mL: 2 g for case 2; the dosage ratio is 100 mL: 4 g for case 3; the dosage ratio is 100 mL: 6 g for case 4; the dosage ratio is 100 mL: 8 g for case 5; the dosage ratio is 100 mL: 10 g for case 6; and the dosage ratio is 100 mL: 12 g for case 7. The dosage ratio of Bacillus subtilis 168 suspension (the rest is the same as step (1)) and the root extract of Stellera chamaejasme is 100 mL: 6 g for case 8.

[0045] Verification of the inhibitory effect of the mixture on the wheat-specific type of Puccinia striae

[0046] (1) Relative inhibition rate of Bacillus subtilis suspension, root extract of Stellera chamaejasme and their mixture against wheat-specific type of Puccinia striata

[0047] The wheat-specific type of Puccinia striae was inoculated on PDA solid culture medium and cultured at 28°C for 48 h. A bacterial cake was punched out from the edge of the colony with a sterile puncher (diameter = 6 mm) and inoculated on the center of a new PDA culture medium plate. 2 μL of the substance to be tested was 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 PDA culture medium plate of the control group grew all over the plate, the growth radius of the pathogen colony was measured, and the inhibition rate was calculated using the following formula.

[0048] Relative inhibition rate = (control colony radius - treated colony radius) / control colony radius × 100%

[0049] (2) Relative inhibition rate of mixtures with different dilution ratios on wheat-specific type of Puccinia striae

[0050] The mixture was diluted 0 times, 10 times, 20 times, 50 times, 100 times and 200 times with sterile water, and the same method as above was used to detect the relative inhibition rate of solutions with different dilution multiples against the wheat-specific type of Puccinia striae, and each treatment was repeated three times.

[0051] Measurement results

[0052] (1) Relative inhibition rate of Bacillus subtilis suspension, root extract of Stellera chamaejasme and their mixture against wheat-specific type of Puccinia striata

[0053] Table 1 Antibacterial effect of each sample prepared by the present invention on wheat-specific type of Puccinia striae

[0054] Substance to be tested Relative antibacterial rate (%) Bacillus subtilis suspension 46.1±2.1％ Stellera chamaejasme Root Extract 63.5±2.9％ Mixture Case 1 75.3±3.8％ Mixture Case 2 88.7±3.5％ Mixture Case 3 92.4±2.8％ Mixture Case 4 94.0±4.6％ Mixture Case 5 93.6±4.2％ Mixture Case 6 90.4±3.6％ Mixture Case 7 83.5±2.8％ Mixture Case 8 68.5±3.6％

[0055] According to the data in Table 1, it can be clearly seen that the mixed case 4 prepared by the present invention has a very significant antibacterial effect on the wheat-specific type of Puccinia striata, with a relative antibacterial rate of up to 94.0%, showing efficient antibacterial ability. The antibacterial effects of mixed case 2-7 on the wheat-specific type of Puccinia striata are all greater than 80%. In addition, the study also found that the Bacillus subtilis suspension prepared by the present invention and the root extract of Stellera chamaejasme used alone have a certain inhibitory effect on the wheat-specific type of Puccinia striata, but the antibacterial rate is not ideal. However, when these two components are mixed in a specific ratio, the antibacterial rate of the obtained mixture is significantly improved. However, the compounding of other Bacillus subtilis and Stellera chamaejasme root extracts did not show a good improvement in antibacterial efficiency.

[0056] (2) Relative inhibition rate of mixtures with different dilution ratios on wheat-specific type of Puccinia striae

[0057] Table 2 Antibacterial effect of mixtures with different dilution ratios (Case 4) on wheat-specific type of Puccinia striae

[0058] Mixtures with different dilution ratios Relative antibacterial rate (%) Original solution 94.0±4.6％ Dilute 10 times solution 91.7±2.1％ Dilute 20 times solution 89.6±4.4％ Dilute 50 times solution 81.0±3.9％ Dilute 100 times solution 69.1±4.3％ Dilute 200 times solution 63.4±2.7％

[0059] According to the data in Table 2, it can be seen that the 10-fold dilution, 20-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 the wheat-specific type of Puccinia striae. Although the antibacterial rate decreases with the increase of the dilution multiple, when the mixture is diluted to 200 times, its antibacterial rate is still above 60%. This result shows that the mixture prepared by the present invention has a strong antibacterial ability and can effectively inhibit the wheat-specific type of Puccinia striae. Therefore, this mixture has a broad application prospect in preventing and controlling wheat stripe rust caused by Puccinia striae.

[0060] (3) Relative inhibition rate of mixtures with different dilution ratios on wheat-specific type of Puccinia striae

[0061] Table 3 Antibacterial effect of mixtures with different dilution ratios (Case 8) on wheat-specific type of Puccinia striae

[0062] Mixtures with different dilution ratios Relative antibacterial rate (%) Original solution 68.5±3.6％ Dilute 10 times solution 62.4±3.5％ Dilute 20 times solution 58.8±3.9％ Dilute 50 times solution 44.7±4.2％ Dilute 100 times solution 35.6±2.8％ Dilute 200 times solution 26.7±3.6％

[0063] According to the data in Table 3, it can be seen that the 10-fold dilution of the mixture (Case 8) prepared by the present invention still has an effect of more than 60%, but the dilutions of 20 times, 50 times, 100 times and 200 times all gradually decrease, and only 26.7% at 200 times. Compared with the mixture (Case 4), the performance of the mixture (Case 8) is poor, and the performance of the dilution is also poor. Even when the mixture (Case 4) is highly diluted to 200 times, it can still maintain an antibacterial rate of more than 60%. When the mixture cases 1-3 and 5-7 are highly diluted to 200 times, they can still maintain an antibacterial rate of about 60%; this fully demonstrates the strong strength and stability of the mixture of the present invention in antibacterial performance. The formula design of the mixture (Case 4) enables the mixture to show efficient inhibitory effects when facing stubborn pathogens such as the wheat-specific type of stripe rust, providing a reliable and lasting solution for the prevention and control of wheat stripe rust. In contrast, although the mixture (Case 8) showed similar antibacterial effects in the initial dilution stage (10 times), its antibacterial ability decreased significantly with the increase in dilution times. When it was diluted 200 times, the antibacterial rate had dropped to 26.7%, showing obvious performance shortcomings. Therefore, the mixture (Case 4) not only showed broad application prospects in the prevention and control of wheat stripe rust with its high antibacterial rate and wide applicability, but also highlighted its dominant position among similar products.

[0064] 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 is prepared by mixing a suspension of Bacillus subtilis and a root extract of Stellera chamaejasme.

2. The liquid microbial composite agent according to claim 1, characterized in that: The dosage ratio of Bacillus subtilis suspension to the root extract of Stellera chamaejasme is 100mL:2-10g.

3. The liquid microbial composite agent according to claim 1, characterized in that: The root extract of Stellera chamaejasme is prepared by a method comprising the following steps: A1. Dry and crush the roots of Stellera chamaejasme; mix the powder with 70-100wt.% ethanol, and soak them to form a preliminary ethanol soaking solution; A2, filtering, separating the soaked material of the root of Stellera chamaejasme, and adding it to 70-100wt.% ethanol for soaking again; the obtained ethanol soaking liquid is mixed with the preliminary ethanol soaking liquid to obtain the target ethanol soaking liquid; A3. Ultrasonic extraction: After filtering, an ethanol extract of the root of Stellera chamaejasme is obtained.

4. The liquid microbial composite agent according to claim 3, characterized in that: In step A1, the solid-liquid ratio of powder to ethanol is 1 g:10-30 mL; and the soaking time is 10-16 hours.

5. The liquid microbial composite agent according to claim 3, characterized in that: In step A2, the solid-liquid ratio of the root soaked material to 70-100 wt.% ethanol is 1 g:3-5 mL.

6. The liquid microbial composite agent according to claim 3, characterized in that: In step A3, the ultrasonic extraction is performed 2-5 times at 25-50° C., the extraction parameters are 200-500W, 20KHZ, and the extraction time for each time is 20-40 minutes.

7. The liquid microbial composite agent according to claim 3, characterized in that: Step A3 is followed by a purification step: A4, filtering and concentrating the ethanol extract under reduced pressure to obtain an ethanol extract; A5, adding the ethanol extract to an ethanol aqueous solution with a concentration of 20-40wt.% to dissolve it to obtain a pre-extraction solution; A6. Extract the product several times continuously with ethyl acetate at room temperature, and mix the obtained extracts; filter and concentrate under reduced pressure to obtain the target extract.

8. The liquid microbial composite agent according to claim 1, characterized in that: The Bacillus subtilis suspension is prepared by a method comprising the following steps: B1. Streak Bacillus subtilis onto LB solid medium and culture at a constant temperature; B2. Pick a single colony and inoculate it into LB medium, and culture it at 35-38°C and 150-250rpm for 16-32h to obtain seed solution; B3. Transfer the seed solution to new LB medium at an inoculum rate of 1%, and culture at 35-38° C. and 150-250 rpm for 16-48 h to obtain a Bacillus subtilis suspension.

9. Use of the liquid microbial composite agent according to any one of claims 1 to 8 in preventing and controlling plant diseases.

10. The use according to claim 9, characterized in that The plant diseases include wheat stripe rust.