Biological control fermentation liquor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological control, and particularly relates to biological control fermentation liquor and a preparation method and application thereof, the preparation method comprises the following steps: S1, raw material treatment: selecting solidago canadensis as a plant raw material, cutting the solidago canadensis into pieces, performing autoclaved sterilization, adding water, and mixing to obtain a mixture; s2, adding the cultured bacillus subtilis and saccharomyces cerevisiae into the mixture in the step S1, mixing and inoculating, and stirring and fermenting to obtain a fermentation stock solution; s3, centrifuging the fermentation stock solution, taking supernate, and filtering to obtain the biological control fermentation liquor. The biological control fermentation liquor prepared by the invention can be used for controlling seed germination, seedling growth and flowering processes of solidago canadensis, and meanwhile, the biological control fermentation liquor can also be used for preparing products for controlling solidago canadensis. The problem that a biological medicament for preventing and treating solidago canadensis is lacked at present is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological control, and specifically relates to a biological control fermentation broth, a preparation method thereof, and an application thereof. Background Art

[0002] Solidago canadensis L. is native to North America and is a perennial herbaceous plant of the Asteraceae family. Due to its high ornamental value and strong adaptability, it was introduced into China as an ornamental plant in the courtyard in the 1930s of the 20th century. However, due to its extremely strong reproductive ability and ecological destructiveness, it has gradually evolved into a malignant invasive species, posing a serious threat to the native ecosystem, agricultural production, and biodiversity in China. Over the years, the control of Solidago canadensis in China has mainly adopted manual control and chemical control methods. Manual control is generally carried out from late August to mid-September every year. However, due to the extremely strong vitality of the underground stems, it is very difficult to achieve a complete removal effect only by manual physical control. Chemical control uses agents such as glufosinate-ammonium and sulfometuron-methyl, which can achieve good control effects. However, chemical control has problems such as environmental pollution and poor environmental safety. Long-term use may even lead to the problem of drug resistance of Solidago canadensis. In addition, a large number of studies have been carried out on using biological control means such as fungi isolated from plants, the parasitic plant Cuscuta japonica, and insect feeding to control Solidago canadensis. However, so far, the biological control technology is not yet mature, and the effect is greatly affected by the environment, and it cannot be prepared and used on a large scale industrially. Summary of the Invention

[0003] Aiming at the problem of the lack of biological control technology for Solidago canadensis, the present invention provides a biological control fermentation broth, a preparation method thereof, and an application thereof. By using the mixed fermentation of Bacillus subtilis and Saccharomyces cerevisiae, the allelochemicals in Solidago canadensis are extracted, and a biological control fermentation broth that can be applied to Solidago canadensis is prepared. The biological control fermentation broth in the present invention has stable properties and can also be applied to other products for controlling Solidago canadensis, solving the problem that the current biological control technology for Solidago canadensis cannot be prepared and used on a large scale industrially.

[0004] The specific technical solutions are as follows:

[0005] A preparation method of a biological control fermentation broth, comprising the following steps:

[0006] S1. Raw material treatment: Select Solidago canadensis as a plant raw material, cut it into pieces and then perform high-pressure sterilization, and add water to mix to obtain a mixture;

[0007] S2. Add the cultured Bacillus subtilis and Saccharomyces cerevisiae to the mixture in step S1 for mixed inoculation, and stir and ferment to obtain a fermentation stock solution;

[0008] S3. Centrifuge the original fermentation broth, take the supernatant, and filter it to obtain the biocontrol fermentation broth.

[0009] For any of the possible implementation manners described above, a further implementation manner is provided. In step S1, the high-pressure sterilization temperature is 121 °C, and the sterilization time is 20 - 30 min; the mass-volume ratio of the plant raw material to water is 1:3 - 7.

[0010] For any of the possible implementation manners described above, a further implementation manner is provided. In step S2, the OD of Bacillus subtilis 600 is 0.8 - 1.5, the OD of Saccharomyces cerevisiae 600 is 1.5 - 2.5, the mixed inoculation amount of Bacillus subtilis and Saccharomyces cerevisiae is 3 - 8%, and the ratio of Bacillus subtilis to Saccharomyces cerevisiae is 1:1 - 2; the fermentation temperature is 30 °C, the fermentation time is 7 d, and the pH value of the fermentation broth during the fermentation process is 5.5 - 6.5. For any of the possible implementation manners described above, a further implementation manner is provided. In step S3, the centrifugation speed is 8000 - 10000 rpm, and the centrifugation time is 10 - 20 min; the filter membrane specification for filtration is 0.22 μm.

[0011] The present invention also provides a biocontrol fermentation broth prepared by the above preparation method.

[0012] The present invention also provides the application of the biocontrol fermentation broth in controlling Solidago canadensis.

[0013] For any of the possible implementation manners described above, a further implementation manner is provided. The application includes directly using the biocontrol fermentation broth and products prepared with the biocontrol fermentation broth, and the products include any one of biocontrol herbicides, bioenzymes, and bio-organic fertilizers.

[0014] For any of the possible implementation manners described above, a further implementation manner is provided. The biocontrol fermentation broth and the products prepared with the biocontrol fermentation broth are applied to the seed germination, and / or seedling growth, and / or flowering stage of Solidago canadensis.

[0015] The beneficial effects of the present invention are:

[0016] 1. The present invention utilizes the method of biological fermentation. Through the mixed fermentation of Bacillus subtilis and Saccharomyces cerevisiae, Bacillus subtilis decomposes complex organic substances (such as starch, cellulose, protein, etc.), and degrades macromolecular substrates into small-molecule glucose by secreting extracellular enzymes. While Saccharomyces cerevisiae utilizes the glucose decomposed by Bacillus subtilis for ethanol fermentation or respiratory metabolism, consuming sugars to reduce the feedback inhibition of the substrate on Bacillus subtilis and promoting the continuous enzyme production of Bacillus. In addition, organic acids (such as lactic acid), vitamins (B group) or amino acids produced by the metabolism of Bacillus can provide growth factors for yeast, and ethanol or CO produced by yeast fermentation may inhibit miscellaneous bacteria, improve the fermentation environment, and indirectly promote the stability of Bacillus. Through the mixed fermentation of the two strains, the fermentation efficiency is enhanced by utilizing their biological metabolic complementarity. In this way, the allelochemicals in Solidago virgaurea can be extracted to the greatest extent, and the autotoxicity of Solidago virgaurea can be improved.

[0017] 2. The present invention uses the allelochemicals carried by Solidago virgaurea itself as the active ingredients for inhibiting the germination, growth and flowering of Solidago virgaurea. Using the preparation method of fermentation extraction, the whole process is safe and pollution-free, and it is a completely biological control agent. And the raw material of the present invention only needs to use Solidago virgaurea, which not only consumes Solidago virgaurea but also solves the problem of raw material source, and the production cost is low. The prepared biological fermentation broth has stable properties, is not restricted by the use environment, and can be used without additives. If long-term preservation is required, only a simple surfactant needs to be added, and there is no chemical pollution, which is suitable for large-scale industrial production and popularization. Description of the Drawings

[0018] Figure 1 The figure shows the experimental results of the inhibition of Solidago virgaurea seed germination by the experimental group and the control group in Experimental Example 1 of the present invention;

[0019] Figure 2 The figure shows the experimental results of the inhibition of Solidago virgaurea seedling growth by the experimental group and the control group in Experimental Example 2 of the present invention;

[0020] Figure 3 The figure shows the experimental results of the physiological effects of Solidago virgaurea seedlings by the experimental group and the control group in Experimental Example 2 of the present invention;

[0021] Figure 4 The figure shows the experimental results of the inhibition of Solidago virgaurea development by the experimental group and the control group in Experimental Example 3 of the present invention. Detailed Embodiments

[0022] The specific embodiments of the present invention will be described in detail below in conjunction with specific drawings. It should be noted that the technical features or combinations of technical features described in the following embodiments should not be considered isolated, and they can be combined with each other to achieve better technical effects.

[0023] Embodiment

[0024] This embodiment provides a method for preparing a biological control fermentation broth, comprising the following steps:

[0025] S1. Raw material treatment: Select Solidago virgaurea as the plant raw material, cut it into pieces respectively and sterilize it under high pressure, and add water and mix to obtain a mixture;

[0026] Specifically, select the above-ground part of Solidago virgaurea as the plant raw material, and the weight of the plant raw material is 100 g. Crush the plant raw material into plant fragments of 2-3 mm, and then place the plant fragments in a high-pressure sterilizer at 121 °C for 20 min. After sterilization, soak it with 500 g of distilled water according to the mass-volume ratio of 1:5.

[0027] S2. Add the cultured Bacillus subtilis and Saccharomyces cerevisiae to the mixture in step S1 for mixed inoculation, and stir and ferment to obtain a fermentation stock solution;

[0028] Specifically, strain culture is carried out first. The culture conditions of Bacillus subtilis are: culture in LB liquid medium with a pH value of 7 at a temperature of 37 °C until OD 600 reaches 1.0 and then set aside. The culture conditions of Saccharomyces cerevisiae are: culture in YPD medium with a pH value of 5.8 at a temperature of 30 °C until OD 600 reaches 2.0 and then set aside. Add the cultured Bacillus subtilis and Saccharomyces cerevisiae to the mixture of plant fragments and water in S1 for inoculation and fermentation, and the inoculation amount is 5%. After inoculation, ferment at 30 °C for 7 days, stir regularly and detect that the pH value of the fermentation broth during the fermentation process is 5.5-6.5. After fermentation is completed, a fermentation stock solution is obtained.

[0029] S3. Centrifuge the fermentation stock solution, take the supernatant and filter it to obtain a biological control fermentation broth.

[0030] Specifically, centrifuge the fermentation stock solution at 8000 rpm for 10 min, take the supernatant, and filter the supernatant under a 0.22 μm membrane to obtain a biological control fermentation broth.

[0031] The biological control fermentation broth prepared in this embodiment is applied to the control of Solidago virgaurea, including inhibiting the seed germination, and / or seedling growth, and / or flowering process of Solidago virgaurea.

[0032] The biocontrol fermentation broth prepared in this example can also be applied to the preparation of products for Solidago virgaurea, including but not limited to biocontrol herbicides, bioenzymes, and bio-organic fertilizers.

[0033] Experimental Example 1: Seed germination inhibition experiment

[0034] Experimental method: The biocontrol fermentation broth prepared in the above example was used as the experimental group, and tribenuron-methyl, Juglans nigra root bark fermentation broth, Ageratum conyzoides fermentation broth, and Humulus scandens fermentation broth were used as the control groups. Among them, tribenuron-methyl was from a commercially available product, and the Juglans nigra root bark fermentation broth, Ageratum conyzoides fermentation broth, and Humulus scandens fermentation broth were prepared according to the fermentation methods of the experimental examples respectively. During the experiment, Solidago virgaurea seeds were placed in a filter paper culture dish (5 mL / dish) containing different concentrations of the experimental group or the control group, 50 seeds per dish, with 3 replicates, and cultured at 25 °C. 5 mL of deionized water and 5 mL of the corresponding medicament were added to the culture dishes in the experimental group and the control group respectively. On the 7th day, the germination rate and radicle length of Solidago virgaurea seeds under different treatments were counted, and 10 seeds were measured for the radicle length of each treatment.

[0035] Experimental results: As shown in the appendix Figure 1 Four plant fermentation broths all had obvious inhibitory effects on the seed germination and radicle growth of Solidago virgaurea (P < 0.05), and the inhibitory degree increased significantly with the increase of the fermentation broth concentration. Generally, the inhibitory effect of the Solidago virgaurea fermentation broth was the best, followed by the Juglans nigra and Ageratum conyzoides fermentation broths, and the inhibitory effect of the Humulus scandens fermentation broth was relatively lower. Under the treatment condition of a low dose of 25 g / L, the four plant fermentation broths could reduce the germination rate of Solidago virgaurea seeds by 19.50% - 33.17%, and the radicle length decreased by 7.19% - 26.14% compared with the blank control; while under the treatment condition of a high dose of 200 g / L, the germination rate and radicle length of Solidago virgaurea seeds decreased by 57.83 - 82.17% and 66.67% - 84.97% respectively. Among them, the inhibitory effect of the 200 g / L Solidago virgaurea fermentation broth on seed germination was equivalent to that of the chemical herbicide tribenuron-methyl.

[0036] Experimental Example 2: Seedling growth inhibition experiment

[0037] Experimental method: The experimental group and the control group were the same as those in Experimental Example 1. During the experiment, the seedlings of Solidago canadensis were transplanted from the field to a spray pot containing a 1:1 mixture of soil and humus. The diameter of the pot was 10 cm, and 3 seedlings were transplanted into each pot. When the seedlings reached a height of 10 cm, watering treatment was carried out. The seedlings of Solidago canadensis were watered with 100 mL of plant fermentation broth or control agent at different concentrations, twice a week for 4 consecutive weeks. After 4 weeks of treatment, the plant height, aboveground biomass (fresh / dry weight), chlorophyll content (SPAD), and MDA content of Solidago canadensis under different treatment conditions were measured. For the plant height of the seedlings, 3 plants were measured for each treatment; after collecting and weighing the aboveground stems and leaves, they were placed in an oven at 50 °C and dried for 7 days to measure the dry weight of the aboveground part. The SPAD value was measured using a handheld chlorophyll meter SPAD-502Plus. For each treatment, the SPAD values of 6 leaves at the same leaf position were measured, and the measurement was repeated 3 times; the determination of MDA content was carried out using a lipid oxidation (MDA) detection kit (Biosharp, BL904A), and the specific steps were referred to the instruction manual.

[0038] Experimental results: As shown in Figure 2 and 3 , the growth rate of the seedlings of Solidago canadensis could be significantly reduced by the four plant fermentation broths. After 4 weeks of treatment, the plant height of the seedlings of Solidago canadensis in the blank control group increased from 10 cm to 18.27 cm. Under the treatment conditions of different concentrations of fermentation broth, the growth of the seedlings of Solidago canadensis became significantly weaker or even stopped growing. The fermentation broth of Solidago canadensis had the best effect, and the fermentation broth of Humulus scandens had the relatively lowest effect. Under the treatment conditions of the low-dose 25 g / L fermentation broth, the seedlings of Solidago canadensis maintained a certain growth rate, and the plant height increased by 4.64 cm - 6.45 cm respectively after 4 weeks of treatment; while under the treatment conditions of the high-dose 200 g / L fermentation broth, the growth of the seedlings of Solidago canadensis significantly slowed down, and there was even no significant change, and the plant height only increased by 0.42 cm - 2.84 cm ( Figure 2 A). The change rules of the fresh weight and dry weight of the aboveground part of the seedlings of Solidago canadensis were similar to those of the plant height. The inhibitory effect of the fermentation broth of Solidago canadensis was the best. Under the treatment conditions of the high-dose 200 g / L fermentation broth, the fresh weight and dry weight of the seedlings of Solidago canadensis were reduced by 44.02% and 45.00% respectively ( Figure 2 B, 2C). The chemical herbicide tribenuron-methyl had a good inhibitory effect on the growth of the seedlings of Solidago canadensis. The inhibitory effect of the 100 g / L - 200 g / L fermentation broth of Solidago canadensis on the growth of the seedlings was equivalent to that of tribenuron-methyl.

[0039] The inhibitory effect of the fermentation broths of four plants on the growth of Solidago canadensis seedlings was directly reflected in the decrease of chlorophyll content in the seedling leaves and the increase of the degree of membrane lipid peroxidation. With the increase of the concentration of the fermentation broths of the four plants, the chlorophyll content in the leaves of Solidago canadensis seedlings decreased sharply, and the decrease was the largest when treated with the fermentation broth of Solidago canadensis. Under the treatment condition of 200 g / L fermentation broth, the chlorophyll content decreased by 31.13%( Figure 3 A). On the contrary, under the treatment condition of the fermentation broth, the MDA content in the seedlings of Solidago canadensis increased significantly. Compared with the blank control, the MDA content increased by up to 6.88 - 9.51 times( Figure 3 B). The effect of 100 g / L fermentation broth of Solidago canadensis on the chlorophyll content in the leaves was equivalent to that of tribenuron-methyl treatment, and the effect of 200 g / L fermentation broth of Solidago canadensis on the degree of membrane lipid peroxidation in the leaves was similar to that of tribenuron-methyl.

[0040] Experimental Example 3: Flowering Inhibition Experiment

[0041] Experimental method: The experimental group and the control group were the same as in Experimental Example 1. During the experiment, the fermentation broth or the control agent was sprayed on the leaf surface of Solidago canadensis plants before flowering, with a spraying volume of 50 mL / plant, sprayed continuously for 3 days, and repeated spraying once after an interval of 7 days. The flowering rate, inflorescence length and pollen viability (TTC staining method) of Solidago canadensis were counted. The flowering rate was the proportion of the open flowers on the inflorescences at specific positions of a single plant to the total flower buds. Three inflorescences were counted for each treatment, and their inflorescence lengths were also counted. The pollen viability was determined using a pollen viability detection kit (Biosharp, product number BL3161A), and the specific steps were referred to the instruction manual. Three counts were made for each treatment.

[0042] Experimental results: As shown in the appendix Figure 4 Under the treatment of the fermentation broths of the four plants, the inflorescence length of Solidago canadensis became significantly shorter, and the flowering rate of the flower buds on the inflorescences decreased significantly. Among them, the fermentation broth of Solidago canadensis had the greatest impact on its flowering. Compared with the blank control, the 200 g / L fermentation broth of Solidago canadensis could reduce the flowering rate by 84.92% and shorten the inflorescence length by 76.00%, and the inhibitory effect was equivalent to that of tribenuron-methyl; the effect of the fermentation broth of Humulus scandens on the flowering rate and inflorescence length of Solidago canadensis was relatively low. Under the treatment condition of 200 g / L fermentation broth, the flowering rate and inflorescence length of Solidago canadensis decreased by 63.49% and 55.24% respectively. The decrease in the flowering rate of Solidago canadensis was related to the decrease in pollen viability. With the increase of the treatment concentration of the fermentation broth, the pollen viability of Solidago canadensis decreased significantly. Under the treatment condition of 200 g / L fermentation broth, compared with the blank control, the pollen viability of Solidago canadensis decreased by 56.47% - 70.82% respectively.

[0043] Although several embodiments of the present invention have been given in this article, those skilled in the art should understand that the embodiments in this article can be changed without departing from the spirit of the present invention. The above embodiments are only exemplary and should not be used to limit the scope of the rights of the present invention by the embodiments in this article.

Claims

1. A method for preparing a biological control fermentation broth, characterized in that, It includes the following steps: S1. Raw material treatment: Select Solidago virgaurea as the plant raw material, cut it into pieces, sterilize it under high pressure, and add water to mix to obtain a mixture; S2. Add the cultured Bacillus subtilis and Saccharomyces cerevisiae to the mixture in step S1 for mixed inoculation, and stir and ferment to obtain a fermentation stock solution; S3. Centrifuge the fermentation stock solution, take the supernatant, and filter it to obtain a biological control fermentation broth.

2. The preparation method of the biocontrol fermentation broth according to claim 1, wherein, In step S1, the high-pressure sterilization temperature is 121 °C, and the sterilization time is 20 - 30 min; the mass-volume ratio of the plant raw material to water is 1:3 - 7.

3. The preparation method of the biocontrol fermentation broth according to claim 1, characterized in that, In step S2, the OD of Bacillus subtilis 600 is 0.8 - 1.5, the OD of Saccharomyces cerevisiae 600 is 1.5 - 2.5, the mixed inoculation amount of Bacillus subtilis and Saccharomyces cerevisiae is 3 - 8%, and the ratio of Bacillus subtilis to Saccharomyces cerevisiae is 1:1 - 2; the fermentation temperature is 30°C, the fermentation time is 7 days, and the pH value of the fermentation broth during the fermentation process is 5.5 - 6.

5.

4. The preparation method of the biological control fermentation broth according to claim 1, characterized in that In step S3, the centrifugation speed is 8000 - 10000 rpm, and the centrifugation time is 10 - 20 min; the filter membrane specification for filtration is 0.22 μm.

5. A biological control fermentation broth obtained by the preparation method according to any one of claims 1 - 4.

6. Use of the biological control fermentation broth according to claim 5 in controlling Solidago virgaurea.

7. Use of the biocontrol fermentation broth according to claim 6 in controlling Solidago canadensis, characterized in that, The use includes directly using the biological control fermentation broth and products prepared using the biological fermentation broth, and the products include any one of a biological control herbicide, a biological enzyme, and a biological organic fertilizer.

8. Use of the biocontrol fermentation broth according to claim 7 in controlling Solidago canadensis, characterized in that, The biological control fermentation broth and the products prepared using the biological fermentation broth are applied to the seed germination, and / or seedling growth, and / or flowering stage of Solidago virgaurea.

Images