A wheat fusarium rust biocontrol agent for inhibiting the growth of Fusarium graminearum and its preparation method and application

Abstract

The present invention belongs to the field of biological control technology, and specifically relates to a preparation technology and application of a wheat fusarium head blight biocontrol agent for inhibiting the growth of Fusarium graminearum. The biocontrol agent of the present invention comprises the following components: 0.3% marine streptomyces gracilis HN43 fermentation crude extract, 1% eugenol, 5% ammonium sulfate, 8% Tween-20 and 20% white carbon. The marine streptomyces gracilis liquid fermentation liquid is extracted by ethyl acetate, the ethyl acetate phase is rotary evaporated, and the fermentation crude extract is obtained by freeze drying, and the fermentation crude extract, eugenol, ammonium sulfate, Tween-20 and white carbon are added to water in proportion to obtain the biocontrol agent. The biocontrol agent of the present invention can significantly inhibit the growth of Fusarium graminearum, and significantly reduce the influence of Fusarium graminearum on the growth and development of wheat, and has the characteristics of being green, environmentally friendly, efficient, stable and not easy to produce drug resistance, and has a wide range of application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and relates to a wheat fusarium wilt biological control preparation for inhibiting the growth of Fusarium graminearum, and a preparation method and application thereof. Background Art

[0002] Fusarium graminearum Fusariumgraminearum ) is an important plant pathogenic fungus that can infect wheat at different growth stages, causing wheat fusarium head blight, stem base rot and other diseases. Under the combined influence of multiple factors such as climate and farming system, the prevalence of wheat fusarium head blight in my country has increased significantly, and is showing a trend of expanding northward and westward. The average annual diseased area in my country exceeds 20% of the wheat planting area. Fusarium graminearum not only causes crop yield loss and quality decline, but also can secrete a variety of fungal toxins such as deoxynivalenol (DON), threatening the health of humans and animals.

[0003] Suspoemulsion is a highly suspended emulsion system formed by dispersing and homogenizing a solid technical drug that is insoluble or slightly soluble in water, an oily liquid technical drug (or an oily liquid obtained by dissolving the technical drug in a small amount of organic solvent), and various additives in an aqueous medium. It has the properties of improving efficacy, expanding the scope of application, and delaying resistance. It is an environmentally friendly dosage form. At the same time, carriers and synergists are added to it to further improve the biological control effect and various stability properties of the spoemulsion.

[0004] For a long time, whether in agricultural production or landscaping management, chemical pesticides have always been the protagonists of disease prevention and control. People rely on chemical pesticides to prevent and control various diseases, resulting in a series of environmental pollution problems, including soil pollution, water pollution, etc. The problem of pesticide residues in agricultural products is relatively serious, posing a health threat to consumers. Using microorganisms to control plant diseases is a green and healthy method. This method is harmless to plants, safe for humans and animals, does not pollute the natural environment, and can reduce the probability of plant diseases. In recent decades, many scientists have conducted research on biological control technology and have achieved many research results. They have screened out many beneficial microorganisms, including fungi, bacteria, and actinomycetes, and biological control methods are constantly being updated and optimized. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a biocontrol preparation for wheat fusarium wilt which can inhibit the growth of Fusarium graminearum.

[0006] The invention also provides a method for preparing a biocontrol preparation for wheat fusarium graminearum which can inhibit the growth of Fusarium graminearum.

[0007] Another object of the present invention is to provide an application of the above-mentioned wheat fusarium rust biocontrol agent in inhibiting the growth of Fusarium graminearum.

[0008] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0009] The invention provides a wheat fusarium scab biocontrol preparation for inhibiting the growth of Fusarium graminearum. The wheat fusarium scab biocontrol preparation comprises the following components by mass fraction: 0.3% crude fermentation extract of Streptomyces marineus HN43, 1% eugenol, 5% ammonium sulfate, 8% Tween-20, 20% white carbon black, and the balance is water.

[0010] The marine actinomycetes used in the present invention are Streptomyces grisea ( Streptomycesmurinus ) HN43, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 10, 2022, with the deposit number: CGMCC NO.25519.

[0011] The present invention also provides a method for preparing a wheat fusarium rust biocontrol agent for inhibiting the growth of Fusarium graminearum, comprising the following steps:

[0012] (1) Marine actinomycete HN43 was inoculated into YMG liquid medium and cultured in a shaking table to obtain seed liquid;

[0013] (2) Inoculate the seed liquid into YMG liquid medium at an inoculum rate of 1.5%, culture on a shaking table, centrifuge to remove the bacteria after the fermentation, collect the supernatant, and obtain the fermentation liquid of Streptomyces cerevisiae HN43; extract the fermentation liquid with an equal volume of ethyl acetate until the ethyl acetate phase turns colorless, rotary evaporate the ethyl acetate phase, and freeze-dry to obtain the crude extract of Streptomyces cerevisiae fermentation;

[0014] (3) Add the crude extract of Streptomyces cerevisiae fermentation, eugenol, ammonium sulfate, Tween-20 and white carbon into water according to the above mass fractions and mix them evenly to obtain the biocontrol agent.

[0015] Furthermore, in steps (1) and (2), the formula of the YMG liquid culture medium is: 10 g malt extract powder, 4 g yeast extract powder, 4 g glucose, 1 L natural seawater, pH 7.2.

[0016] Furthermore, in step (1), the shake flask culture is carried out at a temperature of 28°C and a shaking incubator at 180 rpm for 2 days; and in step (2), the shake flask culture is carried out at a temperature of 28°C and a shaking incubator at 180 rpm for 4 days.

[0017] The present invention also provides an application of the above-mentioned wheat fusarium scab biocontrol agent in inhibiting the growth of Fusarium graminearum.

[0018] The beneficial effects of the present invention are

[0019] (1) The present invention provides a biocontrol agent that can effectively inhibit wheat fusarium head blight caused by Fusarium graminearum, and compared with a single fermentation crude extract, under the synergistic effect of various raw materials, the inhibitory effect on wheat fusarium head blight caused by Fusarium graminearum is improved, and the stability is significantly improved, the operation is simple, and the performance is excellent;

[0020] (2) The biocontrol agent of the present invention can significantly inhibit the growth of Fusarium graminearum and significantly reduce the impact of Fusarium graminearum on the growth and development of wheat. It is green, environmentally friendly, highly effective, stable, and not prone to drug resistance, and has broad application prospects.

[0021] Collection Information

[0022] Deposit date: August 10, 2022;

[0023] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0024] Deposit number: CGMCC NO.25519;

[0025] Address of depository: Beijing;

[0026] Classification name: Streptomyces grisea Streptomycesmurinus . BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The screening results of raw materials for biocontrol agents; wherein (a) is the screening results of carriers for biocontrol agents; (b) is the screening results of dispersants for biocontrol agents;

[0028] Figure 2 The temperature stability results of the biocontrol agents prepared in the examples;

[0029] Figure 3 The pH stability results of the biocontrol agents prepared in the examples are as follows;

[0030] Figure 4 The light exposure time stability results of the biocontrol agents prepared in the examples are shown in FIG. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical means and beneficial effects of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. However, the content of the present invention is not limited to the scope described in the embodiments.

[0032] Example 1: Preparation of crude extract from fermentation of Streptomyces griseus HN43

[0033] Streptomyces natans HN43 is an actinomycete isolated from the soil of mangroves in Haikou. The culture medium used is composed of 10 g malt extract, 4 g glucose, 4 g yeast extract, 1 L natural seawater, pH 7.2. The seed liquid of Streptomyces natans HN43 was inoculated into YMG liquid medium at a 1.5% inoculation rate and cultured at 28°C for 4 days. After the culture, the culture liquid was centrifuged at 8000rpm for 10min to remove the bacteria, and the supernatant was extracted with ethyl acetate until the ethyl acetate phase was colorless. The ethyl acetate phase was collected, filtered with absorbent cotton, and then rotary evaporated and freeze-dried to obtain the final fermentation crude extract of Streptomyces natans HN43.

[0034] Example 2: Preparation of biocontrol agents

[0035] (1) Screening of vectors

[0036] The fermentation crude extract was prepared into a 3 mg / mL solution, and 20% mass fraction of diatomaceous earth, kaolin, bentonite, sodium carboxymethyl nitrocellulose and white carbon were added respectively. After shaking evenly, the mixed solution was allowed to stand for 3 minutes to observe the morphology.

[0037] The results are as follows Figure 1 As shown in (a) (from left to right: diatomaceous earth, kaolin, bentonite, sodium carboxymethyl cellulose nitrate, and white carbon black): diatomaceous earth: the preparation is yellow, with low viscosity, slightly miscible, and obvious stratification after standing; kaolin: the preparation is white-gray, with low viscosity, miscible, and obvious stratification after standing; bentonite: the preparation is white-gray, with high viscosity, miscible, and no obvious stratification after standing; sodium carboxymethyl cellulose nitrate: the preparation is yellow, with high viscosity, immiscible, and obvious stratification after standing; white carbon black: the preparation is white-gray, with low viscosity, miscible, and no obvious stratification after standing. In summary, white carbon black is selected as the carrier of biocontrol preparations.

[0038] (2) Screening of synergists

[0039] The fermentation crude extract was prepared into a 3 mg / mL solution, and 1% eugenol, cinnamaldehyde, allicin and thymol were added respectively. The mixture was thoroughly mixed, and the Oxford cup was placed on the PDA solid culture medium coated with Fusarium graminearum. 200 μL of the mixed solution was added to each Oxford cup. The mixture was cultured at 28°C for 5 days, and the diameter of the inhibition zone was calculated by the cross method.

[0040] The PDA solid medium: 200 g of potatoes were peeled, chopped, boiled in boiling water for 40 min, filtered through 8 layers of gauze, and 20 g of glucose and KH 2 PO 3 3 g, MgSO 4 1.5 g, ddH 2 O to 1 L, natural pH.

[0041] The results are shown in Table 1. Compared with the crude fermentation extract, the diameter of the inhibition zone increased significantly after adding the synergist. After adding eugenol, the diameter of the inhibition zone reached 29.15 mm, so eugenol was selected as the synergist of the biocontrol agent.

[0042] Table 1 Screening of synergists

[0043]

[0044] (3) Screening of emulsifiers

[0045] The fermentation crude extract was prepared into a 3 mg / mL solution, and an equal volume of vegetable oil was added. After stratification, 8% Tween20, Tween40, Tween80 and Span80 were added respectively. After mixing evenly, an equal volume of the mixed solution was centrifuged at 4000 rpm for 2 min. The emulsion stability was calculated based on the emulsion layer volume. Emulsion stability (%) = emulsion layer volume / total volume × 100%

[0046] The results are shown in Table 2. After adding Tween20, the emulsion stability of the emulsion reached 63.22%, and the emulsion stability was good, so Tween20 was finally selected as the emulsifier for the biocontrol agent.

[0047] Table 2 Screening of emulsifiers

[0048]

[0049] (4) Screening of dispersants

[0050] The crude fermentation extract was prepared into a 3 mg / mL solution, and 5% of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, ammonium sulfate and sodium sulfate were added respectively. After mixing evenly, the mixed solution was added with the screened emulsifier and synergist to prepare a suspension. Different suspensions were added into hard water to detect the state of the solution to see whether it immediately dispersed into a cloudy state.

[0051] The results are as follows Figure 1 (b) As shown (from left to right: sodium sulfate, ammonium sulfate, sodium dodecylbenzene sulfonate, sodium dodecyl sulfate): After the suspension containing ammonium sulfate is added to hard water, the solution has no stratification phenomenon and is immediately dispersed into the water with a good dispersion effect, so ammonium sulfate is selected as the dispersant for biocontrol agents.

[0052] Example 3: Verification of the stability of biological control agents in terms of light exposure time, temperature and pH

[0053] (1) Temperature stability

[0054] The biocontrol agents and fermentation crude extracts were placed at 0℃, 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃ for 1 h, cooled to room temperature after treatment, and the antibacterial activity was determined. Three parallel groups were set up. The experimental results are shown in Figure 2 Compared with the crude fermentation extract alone, the biocontrol agent has better temperature stability and still maintains more than 90% of the antibacterial activity after treatment at 90°C.

[0055] (2) pH stability

[0056] The biocontrol agents and fermentation crude extracts were adjusted to pH 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively, and placed at room temperature for 1 h. After the treatment, the pH was adjusted to the original pH of the fermentation liquid, and the antibacterial activity was determined. Three parallel groups were set up. The experimental results are shown in Figure 3 Compared with the crude fermentation extract alone, the biocontrol agent has better resistance to acidic and alkaline environments, better pH stability, and still maintains about 90% of the antibacterial activity when the pH reaches 12.

[0057] (3) Stability of light exposure time

[0058] The biocontrol agents and fermentation crude extracts were placed under light conditions at 28 °C for 1-12 days, and the antibacterial activity was determined in three parallel groups. Figure 4 Compared with the crude fermentation extract alone, the biocontrol agent has better stability in illumination time and still maintains about 93% of the antibacterial activity after 12 days of illumination.

Claims

1. A biocontrol agent for wheat fusarium graminearum that inhibits the growth of Fusarium graminearum, characterized in that: The wheat fusarium rust biocontrol agent comprises the following components by mass fraction: 0.3% Streptomyces griseus ( Streptomyces murinus ) HN43 fermentation crude extract, 1% eugenol, 5% ammonium sulfate, 8% Tween-20, 20% white carbon black, the balance is water; The preparation method of the fermentation crude extract of Streptomyces muridarum HN43 is as follows: inoculating Streptomyces muridarum HN43 into a YMG liquid culture medium, and culturing on a shaking table to obtain a seed liquid; The seed liquid was inoculated into YMG liquid medium at an inoculum rate of 1.5%, and cultured on a shaking table. After the fermentation was completed, the bacteria were removed by centrifugation, and the supernatant was collected to obtain the fermentation liquid of Streptomyces muridarum HN43; the fermentation liquid was extracted with an equal volume of ethyl acetate until the ethyl acetate phase turned colorless, and the ethyl acetate phase was rotary evaporated, and freeze-dried to obtain the crude fermentation extract of Streptomyces muridarum HN43; The YMG liquid culture medium is composed of: 10 g malt extract powder, 4 g glucose, 4 g yeast extract powder, 1 L natural seawater, pH 7.2; The Streptomyces muridarum ( Streptomyces murinus ) HN43, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 10, 2022, with the deposit number: CGMCC NO. 25519.

2. A method for preparing a biocontrol agent for wheat fusarium graminearum that inhibits the growth of Fusarium graminearum as claimed in claim 1, characterized in that: The following steps are involved: (1) Inoculate Streptomyces muridarum HN43 into YMG liquid medium and culture on a shaking table to obtain seed liquid; (2) The seed liquid was inoculated into YMG liquid medium at an inoculum rate of 1.5%, and the culture was carried out on a shaking table. After the fermentation was completed, the bacteria were removed by centrifugation, and the supernatant was collected to obtain the fermentation liquid of Streptomyces muridarum HN43; the fermentation liquid was extracted with an equal volume of ethyl acetate until the ethyl acetate phase turned colorless, and the ethyl acetate phase was rotary evaporated, and the crude extract of the fermentation of Streptomyces muridarum HN43 was obtained after freeze-drying; (3) Add the crude extract of Streptomyces muridarum HN43 fermentation, eugenol, ammonium sulfate, Tween-20 and white carbon black into water according to the above mass fractions and mix them evenly to obtain the biocontrol agent.

3. The preparation method according to claim 2, characterized in that: In step (1), the shake flask culture is carried out at a temperature of 28°C and a shaking incubator at 180 rpm for 2 days; in step (2), the shake flask culture is carried out at a temperature of 28°C and a shaking incubator at 180 rpm for 4 days.

4. Use of the wheat fusarium rust biocontrol agent as claimed in claim 1 in inhibiting the growth of Fusarium graminearum.

Images