CN113832063B - Preparation method and application of Streptomyces faecalis BWL-H1 microbial inoculum - Google Patents

Abstract

The invention provides a preparation method and application of a streptomyces faecalis BWL-H1 microbial agent, and the preparation of the streptomyces faecalis BWL-H1 microbial agent comprises the following steps: s1 Streptomyces faecalis BWL-H1 solid fermentation, S2 preparation wettable powder, the invention selects wheat bran and wormcast to carry out solid fermentation to produce fungus powder, the carrier and auxiliary agent suitable for Streptomyces faecalis BWL-H1 are screened out by biocompatibility determination and national standard method, and the spore content produced by the carrier and auxiliary agent can reach 9.6X10 ⁹ The CFU/g microbial inoculum provided by the invention can inhibit the growth of pathogenic microorganisms, reduce the occurrence of diseases, has a higher control effect on cucumber fusarium wilt, reduces the use of chemical fertilizers and pesticides, and lays a theoretical foundation for further popularization and application.

Description

Preparation method and application of Streptomyces faecalis BWL-H1 inoculant

Technical field

The invention relates to the technical field of microbial fermentation, and in particular to a preparation method and application of Streptomyces faecalis BWL-H1 inoculant.

Background technique

For a long time, pesticides and chemical fertilizers have been very important agricultural production materials and the main way to stabilize or even increase grain production. The abuse of chemical fertilizers and pesticides is widespread throughout the world. It not only wastes resources, but also seriously harms the ecological environment and people's health. Microbial inoculants are an important biological control technology that can replace chemical fertilizers and pesticides. Biocontrol bacteria represented by actinomycetes have been widely used in plant disease control due to their excellent biocontrol effects. China has long carried out research on related biocontrol bacteria preparations, which are used to prevent diseases, protect seedlings and increase production, bringing huge benefits to agricultural production.

Contents of the invention

In view of this, the present invention proposes a preparation method and application of Streptomyces faecalis BWL-H1 inoculant to solve the above problems.

The technical solution of the present invention is achieved as follows: a preparation method of Streptomyces faecalis BWL-H1 inoculant, including the following steps:

S1. Solid fermentation of Streptomyces faecalis BWL-H1: Use vermicompost and wheat bran as the substrate, inoculate Streptomyces faecalis BWL-H1 on the substrate, ferment at 25-30°C for 4-10 days, and dry at a constant temperature of 30-40°C After 20 to 30 hours, crush it and pass it through a 100 to 300 mesh sieve for later use to obtain spore powder;

S2. Preparation of wettable powder: Mix the above spore powder with a wetting agent, a dispersing and suspending agent, and a carrier to prepare a wettable powder, which is Streptomyces faecalis BWL-H1 inoculant. The wettable powder includes the following mass percentage raw materials : Spore powder 8 to 12%, wetting agent 3 to 8%, dispersing and suspending agent 3 to 8%, and the balance is carrier.

Further, the mass ratio of vermicompost and wheat bran in S1 is 2 to 5:1.

Further, the wettable powder in S2 includes the following mass percentage raw materials: 10% spore powder, 5% wetting agent, 5% dispersing and suspending agent, and the balance is carrier.

Further, the wetting agent is one of sodium dodecyl benzene sulfonate, sodium dodecyl sulfate, open powder BX, and AB-3, with a mass concentration of 1 to 5%.

Further, the dispersing and suspending agent is sodium methylene naphthalene sulfonate (NNO) or sodium lignosulfonate, with a mass concentration of 1 to 5%.

Further, the carrier is one of diatomite, kaolin, and white carbon black.

Further, the prepared Streptomyces faecalis BWL-H1 inoculant was used for the control of cucumber fusarium wilt.

Compared with the prior art, the beneficial effects of the present invention are:

The Streptomyces faecalis BWL-H1 strain isolated from soil in the early stage of the present invention grows and reproduces rapidly, produces abundant spores, has strong stress resistance and stable genetic traits. In the present invention, wheat bran and vermicompost are used for solid fermentation to produce bacteria powder with a spore content of up to 9.6×10 ⁹ CFU/g, and then the suitable Streptomyces faecalis BWL-H1 is selected through biocompatibility measurement and national standard methods. The carrier and auxiliaries, namely carrier silica, wetting agent sodium dodecylbenzene sulfonate, dispersing agent NNO, Streptomyces faecalis BWL-H1 spore powder 10%, wetting agent sodium dodecylbenzene sulfonate 5%, dispersant NNO5%, and the rest is supplemented with carrier silica to 100% to make a wettable powder, which has a high control effect on cucumber fusarium wilt.

In the present invention, Streptomyces faecalis BWL-H1 is made into an inoculant, which inhibits the growth of pathogenic microorganisms, reduces the occurrence of diseases, reduces the use of chemical fertilizers and pesticides, and lays a theoretical foundation for further promotion and application.

Description of drawings

Figure 1 Effects of different vectors on the growth of Streptomyces faecalis BWL-H1 colonies;

Figure 2 Effects of different humectants on the growth of Streptomyces faecalis BWL-H1 colonies;

Figure 3 Effects of different dispersing and suspending agents on the growth of Streptomyces faecalis BWL-H1 colonies.

Detailed ways

In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

Unless otherwise specified, the experimental methods used in the examples of the present invention are conventional methods.

Materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources unless otherwise specified.

The Streptomyces strain of the present invention: Streptomyces faecalis BWL-H1, is isolated from the soil under the lychee trees in Bawangling, Changjiang County, Hainan Province, and is stored in the Hainan Green Agricultural Biological Preparation Engineering Research Center.

The cucumber wilt disease of the present invention: Fusarium GF, is isolated from the diseased cucumber species of the Sichuan Academy of Agricultural Sciences and stored in the Hainan Green Agricultural Biological Preparation Engineering Research Center.

Example 1

A preparation method of Streptomyces faecalis BWL-H1 inoculant includes the following steps:

S1. Solid fermentation of Streptomyces faecalis BWL-H1: Use vermicompost and wheat bran as the matrix, the mass ratio of vermicompost and wheat bran is 2:1, inoculate Streptomyces faecalis BWL-H1 on the matrix, and inoculate it at 25°C After fermentation for 4 days and drying at a constant temperature of 30°C for 20 hours, it was crushed and passed through a 100-mesh sieve for later use to obtain spore powder;

S2. Prepare wettable powder: mix 8% of the above spore powder, 3% sodium dodecylbenzene sulfonate, 3% sodium methylene dinaphthalene sulfonate, and 86% silica according to mass percentage to prepare wettable powder. The powder is Streptomyces faecalis BWL-H1.

Example 2

A preparation method of Streptomyces faecalis BWL-H1 inoculant includes the following steps:

S1. Solid fermentation of Streptomyces faecalis BWL-H1: Use vermicompost and wheat bran as the matrix. The mass ratio of vermicompost and wheat bran is 5:1. Streptomyces faecalis BWL-H1 is inoculated on the matrix and incubated at 30°C. After fermentation for 10 days and drying at a constant temperature of 40°C for 30 hours, it was crushed and passed through a 300 mesh sieve for later use to obtain spore powder;

S2. Prepare wettable powder: mix the above spore powder 12%, sodium dodecylbenzene sulfonate 8%, sodium methylene dinaphthalene sulfonate 8%, and silica 72% according to mass percentage. The mass concentration is 1~ 5%, mixed to prepare a wettable powder, which is Streptomyces faecalis BWL-H1.

Example 3

A preparation method of Streptomyces faecalis BWL-H1 inoculant includes the following steps:

S1. Solid fermentation of Streptomyces faecalis BWL-H1: Use vermicompost and wheat bran as the matrix, the mass ratio of vermicompost and wheat bran is 3:1, inoculate Streptomyces faecalis BWL-H1 on the matrix, and inoculate it at 27°C After fermentation for 7 days and drying at a constant temperature of 30-40°C for 24 hours, it was crushed and passed through a 200-mesh sieve for later use to obtain spore powder;

S2. Prepare wettable powder: mix 10% of the above spore powder, 5% sodium dodecylbenzene sulfonate, 5% sodium methylene dinaphthalene sulfonate, and 80% silica according to mass percentage to prepare wettable powder. The powder is Streptomyces faecalis BWL-H1.

Example 4

The difference between this embodiment and fact 3 is that the carrier is diatomite, and the process conditions of the remaining steps remain unchanged.

Example 5

The difference between this embodiment and fact 3 is that the carrier is kaolin, and the process conditions of the remaining steps remain unchanged.

Example 6

The difference between this embodiment and fact 3 is that the wetting agent is sodium lauryl sulfate, and the process conditions of the remaining steps remain unchanged.

Example 7

The difference between this embodiment and Fact 3 is that the wetting agent is BX, and the process conditions of the remaining steps remain unchanged.

Example 8

The difference between this embodiment and fact 3 is that the dispersing and suspending agent is sodium lignosulfonate, and the process conditions of the remaining steps remain unchanged.

Comparative example 1

Streptomyces faecalis BWL-H1 was made into Streptomyces faecalis BWL-H1 according to the method of Zhang Weiwei, Feng Hao et al. ball.

1. Verification experiment

(1) Determination of spore content

The spore powder obtained by fermentation in Examples 1 to 3 was measured for spore content. The results are as follows in Table 1:

It can be seen that the spore content in Implementation 3 can reach 9.6×10 ⁹ CFU/g, indicating that fermentation at a specific temperature and time can produce a larger amount of spores.

(2) Screening of carriers

Add the three carriers of diatomite, kaolin, and silica to the PDA at a concentration of 500 μg/ml, sterilize it at 121°C for 20 minutes, and then pour it into a plate to cool naturally before use.

Culture Streptomyces faecalis BWL-H1 for 7 days, add 2 ml of sterile water to the petri dish, slowly scrape it off with a sterile slide, use a pipette to take a small amount onto the hemocytometer, and count it under a microscope. , after completion, dilute the original solution to the corresponding multiple on the ultra-clean workbench to make a spore suspension with a spore concentration of 10 ³ CFU/mL. Use a pipette to add 100 μL to the PDA plate, and spread evenly with a sterile spreader. Incubate at a constant temperature of 28°C for 5 days, and count the number of colonies. The results are as shown in Table 2.

Table 2 Effects of different vectors on colony growth of Streptomyces faecalis BWL-H1

The research results show that there is no significant difference between the three carriers and ck. However, silica black and diatomaceous earth showed stronger effects on promoting colony formation than kaolin clay (Table 2, Figure 1). In this study, the fineness of the silica is higher and the suspension performance of the product is better. Therefore, in the comparison of Examples 3 to 5, the silica of Example 3 is more suitable as the carrier of Streptomyces faecalis BWL-H1 solid preparation. .

(2) Screening of wetting agents

Taking 2.5g as the base, spore powder accounts for 10%, and when the wetting agent content is 1%, 3%, or 5%, white carbon black is used to complete the content to 100%. Add 100 mL of standard hard water to a 250 mL beaker, and heat the water bath to 25°C. Weigh 2.5g of a uniform sample, pour it all into the beaker at once while keeping the liquid level as stable as possible, and start timing at the same time to record the time it takes for all wetting. Repeat three times and take the average value as the wetting time. The wetting time of the three wetting agents at different concentrations is shown in Table 3.

Table 3 Wetting properties of humectants and their effects on the growth of Streptomyces faecalis BWL-H1 colonies

As can be seen from the above table, SDS has good wettability and can reach 3.83 s at a concentration of 5%. However, due to its greater impact on the germination of actinomycete spores, SDS has poor biocompatibility with this strain. At this concentration, It completely inhibits spore germination, so SDBS is selected as the wetting agent. Adding this wetting agent can make the wetting time less than 2 minutes.

(3) Screening of dispersing and suspending agents

Refer to the method of GB/T 14825-2006 to compare the suspension rates of the two dispersing and suspending agents, Example 3 sodium methylene bisnaphthalene sulfonate (NNO) and Example 8 sodium lignosulfonate, based on 2.5 g, spores When the powder accounts for 10%, the optimal wetting agent accounts for 5%, and the dispersant content is 1%, 3%, and 5% respectively, use white carbon black to complete it to 100%. Weigh an appropriate amount of sample, take a 250mL beaker, add 50mL of standard hard water, heat it to 30±1°C in a constant temperature water bath and then add the sample. Shake the beaker by hand, continue at a gentle shaking rate for 2 minutes, let it stand for 4 minutes, pour the entire amount into a 250mL measuring cylinder, and wash it several times until it reaches full scale. Completely block the opening of the measuring cylinder with a lid, shake it up and down for a period of time and then let it stand in the water bath. At this time, measure the spore concentration as C1. After letting it stand for a long enough time, suck out the supernatant without sucking up the sediment. At this time, measure the spore concentration in the two barrels to be C2. The calculation formula of suspension rate is as follows:

The suspension characteristics of the two dispersing and suspending agents are shown in Table 4.

Table 4 Suspension characteristics of dispersing suspension agents and their effects on the growth of Streptomyces faecalis BWL-H1 colonies

It can be seen from the above table that when no dispersant is added and only 10% of the spore powder is mixed into the silica carrier, the suspension rate of the preparation is only 42.25%. The suspension properties of the dispersant NNO are better than sodium lignosulfonate, and the suspension rate of the preparation can reach 70.97%. Comparing the biocompatibility of the two dispersants with Streptomyces faecalis BWL-H1, NNO did not inhibit the germination of actinomycete spores. Therefore, NNO was selected as a dispersant to be added to the wettable powder.

2. Pot experiment on control of cucumber fusarium wilt disease

Fusarium GF was inoculated into the PDB plate and cultured with shaking in a constant temperature incubator at 28°C for 7 days. After filtering out the hyphae through two layers of cheesecloth, a bacterial suspension with a spore concentration of 1×106 CFU/mL was prepared for later use.

After germination of cucumber seeds, sow them in seedling trays. Two weeks later, transplant them into pots with a diameter of 10cm and a height of 9.5cm. A total of 10 treatments were set up in the test: T1: clear water control; T2: dilute the Streptomyces faecalis BWL-H1 wettable powder of Example 3 100 times for root irrigation. One week later, 100 mL of Fusarium GF spore suspension was used to treat cucumbers. The seedlings were treated with root irrigation; T3: Mix Streptomyces faecalis BWL-H1 microspheres and nutrient soil at a mass ratio of 1:100. Each treatment was repeated three times, with 10 seedlings each time. After 10 days, the disease status of the plants was investigated, and the control effect and disease index standards are shown in Table 5.

Table 5 Different levels of disease classification standards and formulas

The AVERAGE and STDEVP functions in Excel 2007 were used to calculate the mean and standard deviation of the sample, and Waller-Duncan in SPSS 17.0 software was used to perform multiple comparisons (P<0.05). The results are as follows in Table 6.

Table 6 The control efficacy of two preparations of Streptomyces BWL-H1 on cucumber wilt disease

Note: T1, T2, and T3 represent control, wettable powder treatment, and microsphere treatment respectively.

As can be seen from the table above, there are significant differences (P<0.05) between wettable powder treatment, microsphere treatment and control. Streptomyces faecalis BWL-H1 wettable powder has the most obvious control effect on cucumber fusarium wilt, reaching up to 48.65%, which is significantly higher than the control effect of Streptomyces faecalis BWL-H1 microspheres on fusarium wilt.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the present invention. within the scope of protection.

Claims (4)

1. A method for preparing Streptomyces fimicarius BWL-H1 inoculant, which is characterized in that it includes the following steps: S1. Solid fermentation of Streptomyces faecalis BWL-H1: Use vermicompost and wheat bran as the substrate, inoculate Streptomyces faecalis BWL-H1 on the substrate, ferment at 25~30℃ for 4~10 days, and dry at a constant temperature of 30~40℃ After 20 to 30 hours, crush it and pass it through a 100 to 300 mesh sieve for later use to obtain spore powder; S2. Preparation of wettable powder: Mix the above spore powder with a wetting agent, a dispersing and suspending agent, and a carrier to prepare a wettable powder, which is Streptomyces faecalis BWL-H1 inoculant. The wettable powder includes the following mass percentage raw materials : Spore powder 8~12%, wetting agent 3~8%, dispersing and suspending agent 3~8%, the balance is carrier; The wetting agent is sodium dodecylbenzene sulfonate; The dispersing and suspending agent is sodium methylene dinaphthalene sulfonate; The carrier is white carbon black. 2. The preparation method of a kind of Streptomyces faecalis BWL-H1 inoculant according to claim 1, characterized in that: the mass ratio of vermicompost and wheat bran in the S1 is 2~5:1. 3. The preparation method of a kind of Streptomyces faecalis BWL-H1 inoculant as claimed in claim 1, characterized in that: The wettable powder in S2 includes the following mass percentage raw materials: 10% spore powder, 5% wetting agent, 5% dispersing and suspending agent, and the balance is the carrier. 4. Application of the Streptomyces faecalis BWL-H1 inoculant prepared in any one of claims 1 to 3 in the control of cucumber fusarium wilt.