A kind of microbial liquid fertilizer and preparation method thereof

Abstract

The present invention belongs to the technical field of agricultural fertilizers, and specifically relates to a microbial liquid fertilizer and a preparation method thereof. A microbial liquid fertilizer, wherein the microbial liquid fertilizer is formed by mixing a first microbial liquid fertilizer, a second microbial liquid fertilizer and a third microbial liquid fertilizer in a mass ratio of 1-5:1:1-7; wherein the first microbial liquid fertilizer is fermented by Bacillus subtilis; the second microbial liquid fertilizer is fermented by Bacillus licheniformis; and the third microbial liquid fertilizer is fermented by Bacillus velez. The present invention mixes liquid fertilizers fermented by Bacillus subtilis, Bacillus licheniformis and Bacillus velez in a certain mass ratio to prepare a microbial liquid fertilizer with bactericidal and disease-resistant effects, which can improve the control effect on astragalus root rot pathogens, help overcome the continuous cropping obstacles of astragalus, and promote the growth of astragalus plants.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticides, and in particular relates to a microbial liquid fertilizer and a preparation method thereof. Background Art

[0002] Astragalus is a widely used traditional Chinese medicinal material in my country. It is widely planted in my country because of its extremely high application value and economic value. However, continuous cropping obstacles have a great impact on Astragalus during its cultivation. Most of the plots with continuous cropping obstacles are caused by soil-borne diseases. Root rot is one of the more common soil-borne diseases in the cultivation of Astragalus. The pathogens that cause Astragalus root rot mainly include Fusarium oxysporum and Fusarium solani.

[0003] The prevention and control of Astragalus root rot is mainly through screening resistant varieties and using chemical fungicides. Screening resistant varieties is costly and time-consuming, and there is still a lack of Astragalus varieties with strong resistance to root rot. Chemical fungicides are one of the effective means to quickly and effectively prevent and control Astragalus root rot, but long-term application of chemical pesticides is likely to remain in the soil, increasing environmental pressure, and chemical pesticides will also accumulate in the plants, reducing the quality of the medicinal materials.

[0004] Biological control is environmentally friendly and is one of the most promising methods for preventing and controlling soil-borne plant diseases. In the prior art, a variety of microorganisms with control effects on plant diseases have been reported. For example, CN102747020A discloses a strain of Bacillus subtilis that is of great significance for preventing and controlling banana wilt; CN104845906A discloses a strain of Bacillus licheniformis that has great development potential for preventing and controlling grape downy mildew; CN112458012A discloses a strain of Bacillus Velezii that can be used to prevent and control cotton Verticillium wilt, cotton wilt, tomato gray mold and red date black spot.

[0005] In addition, the prior art also discloses that different microorganisms can be compounded for the prevention and control of plant diseases. For example, CN116396899A discloses that a microbial combination of Bacillus subtilis and Bacillus velezii has a certain synergistic effect and can improve the prevention and control effect of cotton Verticillium wilt.

[0006] However, there is no report on combining liquid fertilizers fermented by Bacillus subtilis, Bacillus licheniformis and Bacillus velezii for use as a medicinal fertilizer with bactericidal and disease-resistant effects.

[0007] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0008] The object of the present invention is to provide a microbial liquid fertilizer and a preparation method thereof.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The first object of the present invention is to provide a microbial liquid fertilizer, which is mixed by a first microbial liquid fertilizer, a second microbial liquid fertilizer and a third microbial liquid fertilizer in a mass ratio of 1-5:1:1-7; wherein the first microbial liquid fertilizer is fermented by Bacillus subtilis; the second microbial liquid fertilizer is fermented by Bacillus licheniformis; and the third microbial liquid fertilizer is fermented by Bacillus Velez.

[0011] Preferably, the microbial liquid fertilizer is formed by mixing the first microbial liquid fertilizer, the second microbial liquid fertilizer and the third microbial liquid fertilizer in a mass ratio of 5:1:7.

[0012] More specifically, the Bacillus subtilis was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 11, 2022, with the deposit number: CGMCC No.24391.

[0013] More specifically, the Bacillus licheniformis was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 21, 2022, with the deposit number: CGMCC No.25764.

[0014] More specifically, the Bacillus Velezii was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 21, 2022, with the deposit number: CGMCC No.25765.

[0015] The second object of the present invention is to provide the use of the microbial liquid fertilizer in preventing and controlling Astragalus root rot. The third object of the present invention is to provide a method for preparing the microbial liquid fertilizer, comprising the following steps:

[0016] S1. After streaking and purifying Bacillus subtilis on a plate, a single colony is obtained, and the obtained single colony is inoculated into LB liquid culture medium, and cultured in a constant temperature shaker at 32°C and 180r / min until the OD600 value reaches 0.5, and then the bacterial content of the culture medium is adjusted to 2.5×107 cfu / g with LB liquid culture medium; then, the cultured Bacillus subtilis is inoculated into the fermentation culture medium at an inoculum amount of 3% by mass volume, aerated, and fermented at 32°C for 16h, and the fermentation liquid is collected to obtain the first microbial liquid fertilizer;

[0017] S2. After purifying the Bacillus licheniformis by streaking the plate, a single colony is obtained, and the obtained single colony is inoculated into LB liquid culture medium, and cultured in a constant temperature shaker at 55°C and 180r / min until the OD600 value reaches 0.5, and then the bacterial content of the culture medium is adjusted to 0.8×107 cfu / g with LB liquid culture medium; then the cultured Bacillus licheniformis is inoculated into the fermentation culture medium at an inoculation amount of 3% by mass volume, aerated, and fermented at 55°C for 16h, and the fermentation liquid is collected to obtain the second microbial liquid fertilizer;

[0018] S3. After streaking and purifying the Bacillus Velez subtilis on a plate, a single colony was obtained, and the obtained single colony was inoculated into LB liquid culture medium, and cultured in a constant temperature shaker at 32°C and 180r / min until the OD600 value was 0.5, and then the bacterial content of the culture medium was adjusted to 1.5×107 cfu / g with LB liquid culture medium; then, the cultured Bacillus Velez subtilis was inoculated into the fermentation culture medium at an inoculation amount of 3% by mass volume, aerated, and fermented at 32°C for 16 hours, and the fermentation liquid was collected to obtain the third microbial liquid fertilizer;

[0019] S4. The first microbial liquid fertilizer of step S1, the second microbial liquid fertilizer of step S2 and the third microbial liquid fertilizer of step S3 are mixed in a mass ratio of 1-5:1:7-1 to obtain the microbial liquid fertilizer.

[0020] More specifically, in steps S1-S3, the formula of the LB liquid culture medium is: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and pH 7.0.

[0021] More specifically, the formula of the fermentation medium is: sucrose 20 g / L, trypsin 5 g / L, yeast powder 10 g / L, soybean meal powder 40 g / L, pH value 7.0.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The invention prepares a microbial liquid fertilizer with bactericidal and disease-resistant effects by mixing liquid fertilizers fermented by bacillus subtilis, bacillus licheniformis and bacillus velezii in a certain mass ratio, which can improve the control effect on astragalus root rot pathogens, help overcome the continuous cropping obstacle of astragalus, and promote the growth of astragalus plants.

[0024] Storage Information

[0025] Bacillus subtilis BGB-99R, the deposit date is February 11, 2022, the deposit number is CGMCC No.24391, the preservation center is the General Microbiology Center of China National Culture Collection Administration, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0026] Bacillus licheniformis BGB-84F, the deposit date is September 21, 2022, the deposit number is CGMCC No.25764, the preservation center is the General Microbiology Center of China National Microbiological Culture Collection Administration, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0027] Bacillus velezensis BGB-121R, the deposit date is September 21, 2022, the deposit number is CGMCC No.25765, the preservation center is the General Microbiology Center of China National Microbiological Culture Collection Administration, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solution of the patent of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Embodiment 1:

[0030] Microbial liquid fertilizer

[0031] A method for preparing microbial liquid fertilizer comprises the following steps

[0032] S1. After streaking and purifying Bacillus subtilis on a plate, a single colony is obtained, and the obtained single colony is inoculated into LB liquid culture medium, and cultured in a constant temperature shaker at 32°C and 180r / min until the OD600 value reaches 0.5, and then the bacterial content of the culture medium is adjusted to 2.5×107 cfu / g with LB liquid culture medium; then, the cultured Bacillus subtilis is inoculated into the fermentation culture medium at an inoculum amount of 3% by mass volume, aerated, and fermented at 32°C for 16h, and the fermentation liquid is collected to obtain the first microbial liquid fertilizer;

[0033] S2. After purifying the Bacillus licheniformis by streaking the plate, a single colony is obtained, and the obtained single colony is inoculated into LB liquid culture medium, and cultured in a constant temperature shaker at 55°C and 180r / min until the OD600 value reaches 0.5, and then the bacterial content of the culture medium is adjusted to 0.8×107 cfu / g with LB liquid culture medium; then the cultured Bacillus licheniformis is inoculated into the fermentation culture medium at an inoculation amount of 3% by mass volume, aerated, and fermented at 55°C for 16h, and the fermentation liquid is collected to obtain the second microbial liquid fertilizer;

[0034] S3. After streaking and purifying the Bacillus Velez subtilis on a plate, a single colony was obtained, and the obtained single colony was inoculated into LB liquid culture medium, and cultured in a constant temperature shaker at 32°C and 180r / min until the OD600 value was 0.5, and then the bacterial content of the culture medium was adjusted to 1.5×107 cfu / g with LB liquid culture medium; then, the cultured Bacillus Velez subtilis was inoculated into the fermentation culture medium at an inoculation amount of 3% by mass volume, aerated, and fermented at 32°C for 16 hours, and the fermentation liquid was collected to obtain the third microbial liquid fertilizer;

[0035] S4. Mix the first microbial liquid fertilizer of step S1, the second microbial liquid fertilizer of step S2 and the third microbial liquid fertilizer of step S3 in the mass ratio of Table 1 to obtain the microbial liquid fertilizer. In steps S1-S3, the formula of LB liquid culture medium is: tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, pH value 7.0; the formula of fermentation culture medium is: sucrose 20g / L, tryptone 5g / L, yeast powder 10g / L, soybean meal powder 40g / L, pH value 7.0.

[0036] Table 1 Different microbial liquid fertilizers

[0037] Microbial liquid fertilizer Quality Ratio serial number First microbial liquid fertilizer (A) -- a Second microbial liquid fertilizer (B) -- b The third microbial liquid fertilizer (C) -- c A+B 1:1 d A+C 1:1 e B+C 1:1 f A+B+C 1:1:1 g A+B+C 1:1:7 h A+B+C 5:1:1 i A+B+C 5:1:7 j

[0038] Embodiment 2:

[0039] Effects of different microbial liquid fertilizers on Astragalus root rot

[0040] 1. Test strains

[0041] Astragalus root rot pathogen (Fusarium solani) was obtained by collecting diseased plants in the Astragalus planting area in Gansu Province using conventional tissue separation methods in the laboratory. The Astragalus root rot pathogen was cultured on PDA medium at 25°C for 3 days, propagated and set aside. The formula of PDA medium is: 200g potato, 10g glucose, 20g agar powder, distilled water to 1L, natural pH.

[0042] 2. Experiment with liquid fertilizer

[0043] The microbial liquid fertilizer aj prepared in Example 1 was diluted with sterile water in equal proportions to form 5 mass concentration gradients, wherein the microbial liquid fertilizer was calculated as 100% by mass.

[0044] 3. Test methods

[0045] Add 5 mL of diluted microbial liquid fertilizer to 45 mL of PDA medium cooled to 45°C, mix well and pour into 5 culture dishes with a diameter of 9 cm, and cool to form fertilizer plates. Treat 5 plates with each mass concentration of microbial liquid fertilizer, and set sterile water as a blank control. Use a hole puncher to cut a 5 mm diameter bacterial cake from the edge of the test strain colony after propagation, and inoculate it in the center of the fertilizer plate and the blank control plate, cover the plate and place it in a 25°C constant temperature box for culture. When the diameter of the blank control colony grows to 2 / 3 of the diameter of the culture dish, measure the colony diameter using the cross method, and calculate the inhibition rate of different treatments on the mycelial growth of Astragalus root rot pathogens.

[0046]

[0047] 4. Data Analysis

[0048] DPS software was used for data statistical analysis to obtain the toxicity regression equation and toxicity EC50 value of different treatments. The co-toxicity coefficient CTC of the mixture was calculated according to Sun Yunpei's method, and the synergistic effect was evaluated based on the calculated co-toxicity coefficient CTC. The results are shown in Table 2.

[0049] Microbial liquid fertilizer Virulence regression equation Correlation coefficient r EC50(mg / L) Mixture ATI Mixture TTI Mixture CTC a y=2.3807+1.3210x 0.9840 96.1297 100.0000 -- -- b y=4.2071+0.3928x 0.9687 104.4285 92.0531 -- -- c y=1.2950+1.9896x 0.9960 72.8016 132.0434 -- -- d y=1.0390+1.8608x 0.9777 134.4721 71.4867 96.0266 74.4447 e y=3.2313+0.9721x 0.9781 65.9738 145.7089 116.0217 125.5876 f y=0.2294+2.5149x 0.9648 78.8764 121.8738 112.0483 108.7691 g y=0.1767+3.2274x 0.9676 31.2224 307.8870 108.0322 284.9956 h y=4.1916+0.4613x 0.9757 56.2672 170.8450 124.0397 137.7342 i y=2.5713+1.5078x 0.9594 40.8063 235.5756 103.4424 227.7361 j y=0.7174+3.4622x 0.9866 17.2564 557.0669 116.6428 477.5835

[0050] As shown in Table 2, the first microbial liquid fertilizer, the second microbial liquid fertilizer and the third microbial liquid fertilizer can inhibit the mycelial growth of Astragalus root rot pathogen, and their EC50 are 96.1297 mg / L, 104.4285 mg / L and 72.8016 mg / L respectively.

[0051] When the first microbial liquid fertilizer, the second microbial liquid fertilizer and the third microbial liquid fertilizer were mixed in pairs at a mass ratio of 1:1, the co-toxicity coefficient of microbial liquid fertilizer d on the inhibition of the growth of mycelium of Astragalus root rot pathogen was 74.4447, which was less than 80, showing an antagonistic effect; the co-toxicity coefficient of microbial liquid fertilizer e on the inhibition of the growth of mycelium of Astragalus root rot pathogen was 125.5876, which was greater than 120, showing a synergistic effect; the co-toxicity coefficients of microbial liquid fertilizer f on the inhibition of the growth of mycelium of Astragalus root rot pathogen were 108.7691, between 80-120, showing an additive effect.

[0052] When three different microbial liquid fertilizers were mixed, within the mass ratio of 1-5:1:7-1, the co-toxicity coefficient of the microbial liquid fertilizer on the inhibition of the mycelial growth of Astragalus root rot pathogen was between 137.7342-477.5835, all greater than 120, showing a synergistic effect. Especially when the mass ratio of the three was 5:1:7, the co-toxicity coefficient was 477.5835, and the synergistic effect on the inhibition of the mycelial growth of Astragalus root rot pathogen was the most significant.

[0053] In summary, the microbial liquid fertilizer of the present invention shows an excellent synergistic effect on inhibiting the growth of mycelium of Astragalus root rot pathogen, can improve the control effect on Astragalus root rot pathogen, help overcome the continuous cropping obstacles of Astragalus, and can be used as a medicinal fertilizer with bactericidal and disease-resistant effects.

[0054] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A microbial liquid fertilizer, characterized in that: The microbial liquid fertilizer is formed by mixing a first microbial liquid fertilizer, a second microbial liquid fertilizer and a third microbial liquid fertilizer in a mass ratio of 1-5:1:1-7; wherein the first microbial liquid fertilizer is fermented by Bacillus subtilis; the second microbial liquid fertilizer is fermented by Bacillus licheniformis; the third microbial liquid fertilizer is fermented by Bacillus Velez; wherein, The Bacillus subtilis was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 11, 2022, with the deposit number: CGMCC No.24391; The Bacillus licheniformis was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 21, 2022, with the deposit number: CGMCC No.25764; The Bacillus Velezii was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 21, 2022, with the deposit number: CGMCC No.25765.

2. The microbial liquid fertilizer according to claim 1, characterized in that The microbial liquid fertilizer is formed by mixing a first microbial liquid fertilizer, a second microbial liquid fertilizer and a third microbial liquid fertilizer in a mass ratio of 5:1:

7.

3. Application of the microbial liquid fertilizer according to any one of claims 1 to 2 in preventing and controlling Astragalus root rot.

4. A method for preparing a microbial liquid fertilizer according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. After streaking and purifying the Bacillus subtilis plate, obtain a single colony, inoculate the obtained single colony into LB liquid culture medium, and culture it in a constant temperature shaker at 32°C and 180r / min until the OD 600 The value was 0.5, and then the bacterial content of the culture medium was adjusted to 2.5×10 7 cfu / g; then, the cultured Bacillus subtilis was inoculated into the fermentation culture medium at an inoculation rate of 3% by mass volume, aerated, fermented at 32°C for 16h, and the fermentation liquid was collected to obtain the first microbial liquid fertilizer; S2. After streaking and purifying the Bacillus licheniformis, a single colony was obtained, and the obtained single colony was inoculated into LB liquid culture medium, and cultured in a constant temperature shaker at 55°C and 180r / min until OD 600 The value was 0.5, and then the bacterial content of the culture medium was adjusted to 0.8×10 7 cfu / g; then, the cultured Bacillus licheniformis was inoculated into the fermentation culture medium at an inoculation rate of 3% by mass volume, aerated, fermented at 55°C for 16h, and the fermentation liquid was collected to obtain a second microbial liquid fertilizer; S3. After streaking and purifying Bacillus velezensis, obtain a single colony, inoculate the obtained single colony into LB liquid culture medium, and culture in a constant temperature shaker at 32°C and 180r / min until OD 600 The value was 0.5, and then the bacterial content of the culture medium was adjusted to 1.5×10 7 cfu / g; then, the cultured Bacillus Velezii was inoculated into the fermentation culture medium at an inoculation rate of 3% by mass volume, aerated, fermented at 32°C for 16h, and the fermentation liquid was collected to obtain a third microbial liquid fertilizer; S4. The first microbial liquid fertilizer of step S1, the second microbial liquid fertilizer of step S2 and the third microbial liquid fertilizer of step S3 are mixed in a mass ratio of 1-5:1:7-1 to obtain the microbial liquid fertilizer.

5. The preparation method according to claim 4, characterized in that: In steps S1-S3, the formula of LB liquid culture medium is: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and pH 7.

0.

6. The preparation method according to claim 4, characterized in that: In steps S1-S3, the formula of the fermentation culture medium is: sucrose 20g / L, tryptone 5g / L, yeast powder 10g / L, soybean meal powder 40g / L, pH value 7.0.