Biological herbicide for preventing and treating solidago canadensis as well as preparation method and use method of biological herbicide

Abstract

The invention belongs to the technical field of biological prevention and treatment, and particularly relates to a biological herbicide for preventing and treating solidago canadensis and a preparation and use method thereof.The biological herbicide is prepared from, by volume, 94%-97% of biological fermentation liquor, 0.75%-1% of Tween 80, 1.5%-2% of paraffin oil, 0.1%-0.2% of xanthan gum and 1%-2% of azone. The biological fermentation liquid is prepared by fermenting solidago canadensis. 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 high-pressure sterilization, and adding water for 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, filtering to obtain biological fermentation liquor, adding Tween 80, paraffin oil, xanthan gum and azone into the biological fermentation liquor at one time, stirring until the materials are completely dissolved, adjusting the pH value, and filtering to remove impurities to obtain the biological herbicide. The biological herbicide for preventing and treating the solidago canadensis can be used for preventing and treating seed germination, seedling growth and flowering processes of the solidago canadensis, and solves the problem that a biological medicament for preventing and treating the solidago canadensis is lacked at present.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological control, and particularly relates to a biological herbicide for controlling Solidago canadensis and its preparation and use methods. 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. 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 eradication effect by simply using manual physical control. Chemical control uses herbicides 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 in industrial production. 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 herbicide for controlling Solidago canadensis and its preparation and use methods. By using the mixed fermentation of Bacillus subtilis and Saccharomyces cerevisiae, the allelochemicals in Solidago canadensis are extracted, and a biological herbicide for controlling Solidago canadensis based on Solidago canadensis is prepared. The biological herbicide for controlling Solidago canadensis 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 in industrial production.

[0004] The specific technical solution is as follows:

[0005] A biological herbicide for controlling Solidago canadensis comprises the following components by volume fraction: 94.8 - 96.5% biological fermentation broth, 0.75 - 1% Tween 80, 1.5 - 2% paraffin oil, 0.1 - 0.2% xanthan gum, and 1 - 2% azone, and the biological fermentation broth is prepared by fermenting Solidago canadensis.

[0006] For any of the possible implementation manners described above, a further implementation manner is provided. The biological herbicide comprises the following components by volume fraction: 96.65% biological fermentation broth, 0.75% Tween 80, 1.5% paraffin oil, 0.1% xanthan gum, and 1% azone.

[0007] For any of the possible implementation manners described above, a further implementation manner is provided. The pH value of the herbicide is 5.5 - 6.5.

[0008] A preparation method of a biological herbicide for controlling Solidago virgaurea includes the following steps:

[0009] S1. Raw material treatment: Select Solidago virgaurea as the plant raw material, cut it into pieces, perform high-pressure sterilization, and add water to mix to obtain a mixture;

[0010] S2. Add the cultured Bacillus subtilis and Saccharomyces cerevisiae into the mixture in step S1 for mixed inoculation, and stir and ferment to obtain a fermentation stock solution;

[0011] S3. Centrifuge the fermentation stock solution, take the supernatant, filter it to obtain a biological fermentation broth, add Tween 80, paraffin oil, xanthan gum, and azone to the biological fermentation broth at one time, stir until completely dissolved, adjust the pH, and filter to remove impurities to obtain the biological herbicide.

[0012] 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 1:5.

[0013] 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.

[0014] For any of the possible implementation manners described above, a further implementation manner is provided. In step S3, the centrifugation speed is 8000 rpm, and the centrifugation time is 10 min; the filter membrane specification for filtration is 0.22 μm.

[0015] The present invention also provides a usage method of the biological herbicide for controlling Solidago virgaurea, including the following steps: Spray before Solidago virgaurea blossoms, spray continuously for 3 days first, and repeat spraying once after an interval of 7 days.

[0016] For any of the possible implementation manners described above, a further implementation manner is provided. The spraying amount for each spraying is 80 - 100 L / mu.

[0017] The beneficial effects of the present invention are as follows:

[0018] 1. In the present invention, Bacillus subtilis and Saccharomyces cerevisiae are mixed and fermented, and their biological metabolic complementarity is utilized to improve the fermentation efficiency, obtaining a mixed fermentation broth rich in various allelochemicals. By adding different additives to the mixed fermentation broth, the stability and activity of the fermentation broth can be significantly improved. Tween 80, as a non-ionic surfactant, significantly reduces the surface tension of the fermentation broth, enhances the wettability and spreading property, and promotes the penetration of the active ingredients through the epidermis of Solidago canadensis; paraffin oil can dissolve the epidermal wax layer of Solidago canadensis, enhance the permeability of the fermentation broth, and at the same time, as a carrier, prolong the retention time of the fermentation broth on the epidermis, reducing the photolysis and volatilization losses; xanthan gum, as a thickener and stabilizer, can improve the adhesion of the fermentation broth on the inclined leaf surface, increase the leaf surface deposition amount, and reduce the droplet drift; azone can significantly destroy the cuticular lipid structure of Solidago canadensis, enhance the cell membrane permeability, promote transmembrane absorption, and improve the inhibitory effect. After adding the above 4 additives, the control effect of the biological herbicide on Solidago canadensis is significantly higher than that of the biological fermentation broth.

[0019] 2. The present invention uses the allelochemicals carried by Solidago canadensis itself as the active ingredients to inhibit the germination, growth and flowering of Solidago canadensis. The preparation method by fermentation extraction is safe and pollution-free throughout the process, and it is a completely biological control agent. Moreover, the raw materials of the present invention need to use Solidago canadensis, which not only consumes Solidago canadensis but also solves the problem of raw material source, and the production cost is low. The biological herbicide prepared by the present invention has stable properties, is not restricted by the use environment, has no chemical pollution, and is suitable for large-scale industrial production and popularization. Description of the Drawings

[0020] Figure 1 The figure shows the experimental results of the inhibition of Solidago canadensis seed germination by the experimental group and the control group in Experimental Example 1 of the present invention;

[0021] Figure 2 The figure shows the experimental results of the inhibition of Solidago canadensis seedling growth by the experimental group and the control group in Experimental Example 2 of the present invention;

[0022] Figure 3 The figure shows the experimental results of the physiological effects of Solidago canadensis seedlings by the experimental group and the control group in Experimental Example 2 of the present invention;

[0023] Figure 4 The figure shows the experimental results of the inhibition of Solidago canadensis development by the experimental group and the control group in Experimental Example 3 of the present invention. Detailed Embodiments

[0024] 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 described in the following embodiments or the combination of technical features should not be considered isolated, and they can be combined with each other to achieve better technical effects.

[0025] Embodiment

[0026] This embodiment provides a biological herbicide for controlling Solidago canadensis, which comprises the following components by volume fraction: 96.65% biological fermentation broth, 0.75% Tween 80, 1.5% paraffin oil, 0.1% xanthan gum and 1% azone.

[0027] This embodiment also provides a preparation method of a biological herbicide for controlling Solidago canadensis, which comprises the following steps:

[0028] S1. Raw material treatment: Select Solidago canadensis as the plant raw material, cut it into pieces respectively and sterilize it under high pressure, and add water to mix to obtain a mixture;

[0029] Specifically, select the above-ground part of Solidago canadensis 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.

[0030] S2. Add the cultured Bacillus subtilis and Saccharomyces cerevisiae into the mixture in step S1 for mixed inoculation, and stir and ferment to obtain a fermentation stock solution;

[0031] Specifically, strain cultivation is carried out first. The cultivation conditions of Bacillus subtilis are: cultivate in LB liquid medium with a pH value of 7 at a temperature of 37 °C until the OD 600 reaches 1.0 and then reserve. The cultivation conditions of Saccharomyces cerevisiae are: cultivate in YPD culture medium with a pH value of 5.8 at a temperature of 30 °C until the OD 600 reaches 2.0 and then reserve. Add the cultured Bacillus subtilis and Saccharomyces cerevisiae into 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.

[0032] S3. Centrifuge the fermentation stock solution, take the supernatant and filter it to obtain a biological fermentation broth, add Tween 80, paraffin oil, xanthan gum and azone to the biological fermentation broth at one time, stir until completely dissolved, adjust the pH, and filter to remove impurities to obtain a biological herbicide.

[0033] Specifically, the fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant was taken. The supernatant was filtered through a 0.22-μm membrane to obtain the biological fermentation broth. The concentration of the biological fermentation broth was diluted to 200 g / L, and Tween 80, paraffin oil, xanthan gum, and azone were sequentially added to the fermentation broth and stirred until completely dissolved. The pH was adjusted to 5.5 - 6.5, and after filtering to remove impurities, it was stored at 4°C for standby.

[0034] This example provides a method for using a biological herbicide for controlling Solidago canadensis. Specifically, it is sprayed before Solidago canadensis blooms. First, it is continuously sprayed for 3 days, and after a 7-day interval, it is sprayed again. Before blooming includes the seed germination of Solidago canadensis, and / or the seedling growth, and / or the stage waiting to bloom. The spraying amount per time is 80 - 100 L / acre.

[0035] Experimental Example 1: Seed germination inhibition experiment

[0036] Experimental method: Using the biological herbicide for controlling Solidago canadensis prepared in the above example as the experimental group, and the biological fermentation broth as the control group. During the experiment, Solidago canadensis seeds were placed in a filter paper culture dish (5 mL / dish) containing 200 g / L biological fermentation broth (SFB) or biological herbicide (CSBH), 50 seeds per dish, with 3 replicates, and cultured at 25°C, with a water control set. On the 7th day, the germination rate and radicle length of Solidago canadensis seeds among different treatments were counted. For the radicle length, 10 seeds were measured for each treatment.

[0037] Experimental results: As shown in the appendix Figure 1 shown, both SFB and CSBH had obvious inhibitory effects on the seed germination and radicle growth of Solidago canadensis (P < 0.05). Both of them reduced the germination rate of Solidago canadensis seeds to less than 18%, and the radicle length was shortened to within 0.23 cm (appendix Figure 1 -B). Compared with SFB, CSBH had a stronger inhibitory effect on the seed germination of Solidago canadensis (appendix Figure 1 -A).

[0038] Experimental Example 2: Seedling growth inhibition experiment

[0039] Experimental method: When the seedlings of Solidago virgaurea grow to a plant height of 10 cm, watering treatment is carried out. The seedlings of Solidago virgaurea are watered with 100 mL of 200 g / L SFB or CSBH twice a week for 4 consecutive weeks. After 4 weeks of treatment, the plant height, fresh weight and dry weight of the above-ground part, chlorophyll content (SPAD) and MDA content of the seedlings of Solidago virgaurea are measured respectively. The SPAD value is measured by a handheld chlorophyll meter SPAD-502Plus. The SPAD values of 6 leaves at the same leaf position are measured for each treatment, and the measurement is repeated 3 times; the determination of MDA content is carried out using a lipid oxidation (MDA) detection kit (Biosharp, BL904A), and the specific steps refer to the instruction manual.

[0040] Experimental results: As shown in Figure 2 and 3 , both SFB and CSBH can significantly reduce the growth rate of the seedlings of Solidago virgaurea. After 4 weeks of treatment, the plant height of the seedlings of Solidago virgaurea in the blank control group increased significantly to 20.33 cm, while under the treatment conditions of SFB and CSBH, the seedlings of Solidago virgaurea basically stopped growing, and the plant heights of the seedlings of Solidago virgaurea in the SFB and CSBH treatment groups increased by 1.69 cm and 0.22 cm respectively (see Figure 2 A). The change rules of the fresh weight and dry weight of the above-ground part are similar to those of the plant height. Compared with the blank control, the fresh weight and dry weight of the seedlings of Solidago virgaurea treated with SFB decreased by 43.11% and 44.58% respectively, while those treated with CSBH decreased by 56.55% and 57.54% respectively (see Figure 2 B, 2C).

[0041] The growth inhibitory effects of SFB and CSBH on the seedlings of Solidago virgaurea are directly reflected in the decrease of the chlorophyll content in the seedling leaves and the increase of the degree of membrane lipid peroxidation. Under the treatment conditions of SFB and CSBH, the chlorophyll content decreased by 26.73% and 37.15% respectively (see Figure 3 A). On the contrary, the MDA content of the seedlings of Solidago virgaurea increased significantly. Compared with the blank control, the MDA content increased by up to 7.54 and 9.62 times at most (see Figure 3 B).

[0042] Experimental Example 3: Flowering inhibition experiment

[0043] 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 the Solidago virgaurea plants before flowering. The spraying volume was 50 mL / plant, and it was continuously sprayed for 3 days. After an interval of 7 days, it was sprayed again once, and the spraying volume was 100 L / mu. The flowering rate, inflorescence length, and pollen viability (TTC staining method) of Solidago virgaurea were statistically analyzed. The flowering rate was the proportion of the opened flowers on the inflorescences at specific positions of a single plant to the total flower buds. Three inflorescences were statistically analyzed for each treatment, and their inflorescence lengths were also statistically analyzed. The pollen viability was determined using a pollen viability detection kit (Biosharp, BL3161A), and the specific steps were referred to the instruction manual. Each treatment was statistically analyzed 3 times.

[0044] Experimental results: As shown in the Figure 4 appendix, under the treatments of SFB and CSBH, the inflorescence lengths of Solidago virgaurea were significantly shortened, and the flowering rates of the flower buds on the inflorescences were significantly reduced. Among them, the flowering rates were reduced to 17.78% and 9.55% respectively, and the inflorescence lengths were shortened by 70.11% and 79.06% ( Figure 4 A, B). The decrease in the flowering rate of Solidago virgaurea was related to the decrease in pollen viability. Under the treatments of SFB and CSBH, the pollen viability of Solidago virgaurea was significantly reduced. Compared with the blank control, the pollen viability of Solidago virgaurea was reduced by 63.89% and 76.17% respectively ( Figure 4 C).

[0045] 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 as the limitation of the scope of the rights of the present invention.

Claims

1. A biological herbicide for controlling Solidago canadensis, characterized in that, It comprises the following components by volume fraction: 94 - 97% of biological fermentation broth, 0.75 - 1% of Tween 80, 1.5 - 2% of paraffin oil, 0.1 - 0.2% of xanthan gum and 1 - 2% of azone, and the biological fermentation broth is prepared by fermenting Solidago virgaurea.

2. The biological herbicide for controlling Solidago canadensis according to claim 1, characterized in that, The biological herbicide comprises the following components by volume fraction: 96.65% of biological fermentation broth, 0.75% of Tween 80, 1.5% of paraffin oil, 0.1% of xanthan gum and 1% of azone.

3. A biological herbicide for controlling Solidago canadensis according to claim 1, characterized in that, The pH value of the herbicide is 5.5 - 6.

5.

4. A method for preparing a biological herbicide for controlling Solidago canadensis L. according to any one of claims 1 to 3, characterized in that, It comprises the following steps: S1. Raw material treatment: Select Solidago virgaurea as the plant raw material, cut it into pieces and sterilize it under high pressure, then add water and mix to obtain a mixture. S2. Add the cultured Bacillus subtilis and Saccharomyces cerevisiae into the mixture in step S1 for mixed inoculation, and stir and ferment to obtain the original fermentation broth. S3. Centrifuge the original fermentation broth, take the supernatant and filter it to obtain the biological fermentation broth. Add Tween 80, paraffin oil, xanthan gum and azone to the biological fermentation broth at one time, stir until completely dissolved, adjust the pH, and filter to remove impurities to obtain the biological herbicide.

5. The preparation method of a biological herbicide for controlling Solidago canadensis described in claim 4, characterized in that, 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.

6. The preparation method of a biological herbicide for controlling Solidago canadensis described in claim 4, 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 7d, and the pH value of the fermentation broth during the fermentation process is 5.5 - 6.

5.

7. The preparation method of a biological herbicide for controlling Solidago canadensis L. according to claim 4, 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.

8. A method for using a biological herbicide for controlling Solidago canadensis L. obtained by the preparation method according to any one of claims 1 to 3 or any one of claims 4 to 7, characterized in that, It comprises the following steps: Spray before Solidago virgaurea blossoms. First, spray continuously for 3 days, and repeat spraying once after an interval of 7 days.

9. A method for using a biological herbicide for controlling Solidago canadensis, characterized in that, The spraying amount for each spraying is 80 - 100 L / acre.

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