Algae-inhibiting agent for preventing and controlling strong anterior ditch algae in stichopus japonicus circle and application of algae-inhibiting agent

Abstract

According to the algistat, the whole overground part of Artemisia annua is dried at 60 DEG C and crushed to 150 meshes, tea seed cake powder is crushed to 150-mesh and 80-mesh trichoderma harzianum leavening is used as raw materials, and the algistat is obtained by uniformly mixing the raw materials and the trichoderma harzianum leavening and the trichoderma harzianum leavening and the trichoderma harzianum leavening and the trichoderma harzianum leavening and the trichoderma harzianum leavening and the trichoderma harzianum leavening and the trichoderma harzianum leavening. The preparation method of the algicide for the premnant algae in the stichopus japonicus circle is simple, the raw materials are wide in source, low in cost and high in algicide inhibition efficiency, outbreak of the premnant algae can be controlled, harm of toxin released by the premnant algae to stichopus japonicus is reduced, resistance is not prone to being generated, and the algicide has no toxic or side effect on the stichopus japonicus and no harm to the environment and is worthy of popularization and application. The application effect is obvious, and the market prospect is wide.

Description

Technical Field

[0001] The invention belongs to the field of water purification in marine aquaculture, relates to an algae inhibitor for controlling strong anterior algae in sea cucumber circles and an application thereof, and specifically relates to an algae inhibitor for controlling strong anterior algae in sea cucumber circles formed by combining plants, economic crop byproducts and microorganisms and an application thereof. Background Art

[0002] Amphidinium carterae Hulburt is one of the harmful red tide dinoflagellates. It first appeared in the southern seas of China. In recent years, it has gradually spread to the northern seas of China and entered the aquaculture waters. Sea cucumbers are an important aquaculture species in the Liaoning coast of northern China, with high economic value. The investigation found that due to the biological characteristics of sea cucumbers and the corresponding water quality requirements, Amphidinium carterae Hulburt is very easy to break out in the sea cucumber aquaculture waters. In recent years, the harm of Amphidinium carterae Hulburt to sea cucumber aquaculture has gradually emerged. Because Amphidinium carterae Hulburt secretes toxic substances, it will have a great adverse effect on the growth of sea cucumbers during its outbreak period. In severe cases, it will cause large-scale deaths, causing huge economic losses to the sea cucumber aquaculture industry.

[0003] At present, the prevention and control methods for Dialella robusta in sea cucumber aquaculture water bodies are mainly herbicides and chemical disinfectants, which have toxic side effects on sea cucumbers, pollute the environment and cause food safety hazards. The sea cucumber aquaculture water surface in my country is very large, and the losses caused by Dialella robusta every year are also huge. Therefore, it is urgent to find a more environmentally friendly and efficient algae inhibitor for the prevention and control of Dialella robusta in sea cucumber aquaculture, so as to control the harmful algae Dialella robusta in sea cucumber aquaculture, protect the ecological environment of water bodies, and promote the healthy development of sea cucumber aquaculture. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an algae inhibitor for controlling the strong anterior channel algae in the sea cucumber circle and its application. The algae inhibitor is formed by compounding plants, economic crop by-products and microorganisms. It is simple to prepare, has high algae inhibition efficiency, is not easy to produce resistance, has no toxic side effects on sea cucumbers, and is harmless to the environment.

[0005] The technical solution of the present invention is: An algae inhibitor for preventing and controlling sea cucumber circle strong front groove algae, the special feature of which is that the algae inhibitor is a mixture of artemisia annua, tea seed cake and Trichoderma harzianum.

[0006] Furthermore, in terms of weight, 0.03 parts of Artemisia annua, 0.05-0.1 parts of tea seed cake, and 0.10-0.15 parts of Trichoderma harzianum are used.

[0007] Furthermore, according to the weight proportions, 0.03 parts of Artemisia annua, 0.1 parts of tea seed cake, and 0.15 parts of Trichoderma harzianum are used at a dosage of 0.28 mg / mL, and the algae inhibition rate is 99%-100% in 3-6 days.

[0008] Furthermore, according to the weight proportions, 0.03 parts of Artemisia annua, 0.05 parts of tea seed cake, and 0.10 parts of Trichoderma harzianum are used at a dosage of 0.18 mg / mL, and the algae inhibition rate is 96%-98% in 3-6 days.

[0009] Further, the preparation process is as follows: The whole plant of the aerial part of Artemisia annua was dried at 60°C and crushed to 150 mesh, tea seed cake was crushed to 150 mesh, and the fermentation product of Trichoderma harzianum was evenly mixed with 80 mesh to obtain the sea cucumber circle strong front channel algae inhibitor.

[0010] Further, the preparation process of Trichoderma harzianum fermentation product is as follows: According to the percentage by weight, 1% cornmeal, 2% rice bran, 1% soybean meal, 1% sucrose, and the rest water are used as a culture medium, and 4% of the volume of the culture medium is inoculated with Trichoderma harzianum fermentation seed liquid, and cultured for 72 hours under the conditions of pH=6.5, 30°C, and 200r / min to obtain a liquid fermentation liquid; A carrier was added at a mass volume ratio of 1:1 g / mL to the liquid fermentation liquid. The carrier was zeolite powder and rice husk powder at a mass ratio of 4:6. The mixture was adsorbed, dried, and crushed to 80 meshes to obtain a Trichoderma harzianum fermentation product.

[0011] An application of an algae inhibitor for controlling the robust algae in the sea cucumber circle in inhibiting the robust algae.

[0012] Furthermore, the algaecide is used at a concentration of 0.18-0.28 mg / mL.

[0013] Furthermore, the inhibition rate of the strong algae inhibitor at a concentration of 0.18-0.28 mg / mL on strong algae for 72 hours was 95%-99%.

[0014] The beneficial effects of the present invention are: (1) The compound anti-algae effect is strong. The plant is Artemisia annua, the plant byproduct is tea seed cake, and the fungus is Trichoderma harzianum, all of which have strong anti-algae effects. The compound anti-algae agent can act on multiple targets at the same time, improving the anti-algae effect and not easily causing algae to develop drug resistance. In addition, Artemisia annua can promote the growth of sea cucumbers and improve immunity to a certain extent, Trichoderma harzianum is a commonly used agricultural probiotic, and tea seed cake has a killing effect on fish at a certain concentration, but has no inhibitory effect on sea cucumbers.

[0015] (2) The raw materials are widely available and low in cost. Artemisia annua is a traditional Chinese medicinal herb for extracting artemisinin. It has abundant wild resources, which reduces the cost of use. Tea seed cake is a byproduct of common oil crops. It has a stable source, is cheap, and contains saponins that have an anti-algae effect. Trichoderma harzianum is easy to obtain, has strong adaptability, and has a simple fermentation process, which further reduces production costs.

[0016] (3) Environmentally friendly and safe. The compounded algaecide is a combination of degradable natural products and will not cause pollution to the environment after use.

[0017] (4) High algae inhibition efficiency. Under the specific combination of the present invention, the dosage at a relatively low concentration (such as 0.28 mg / mL, 0.18 mg / mL) can achieve an algae inhibition rate of more than 90% in 3 days, which is highly effective in inhibiting algae and can inhibit the outbreak of the robust angiospermum and reduce the harm of its toxin release to sea cucumbers.

[0018] (5) No side effects on sea cucumber farming. The use of Artemisia annua, tea seed cake powder and Trichoderma harzianum fermentation product at low concentrations in the present invention has no toxic side effects on sea cucumber farming, ensuring the safety of use.

[0019] In summary, the preparation method of the sea cucumber circle strong anterior channel algae inhibitor provided by the present invention is simple. The algae inhibitor is formed by compounding plants, economic crop by-products and microorganisms. The raw material source is wide and the cost is low. The algae inhibition efficiency is high, it is not easy to produce resistance, it has no toxic side effects on sea cucumbers, and it is harmless to the environment. It has significant application effects and broad market prospects. DETAILED DESCRIPTION

[0020] Example 1 (1) Purification and fermentation of Trichoderma harzianum Weigh 10 mg of commercially available Trichoderma harzianum powder, dissolve it in 10 mL of sterile distilled water, shake and mix, use an inoculation loop to streak a small amount on a PDA agar plate, and incubate at 30°C. After 1 to 2 days, colonies grow, and according to the characteristics and color of the colonies, combined with microscopic observation, pick a typical single colony (picking criteria: the single colony is flat and irregular in shape, hyphae can be seen with the naked eye, and the colony is dark green). Continue to purify twice, pick a typical colony and place it in 100 mL of PDA liquid culture medium, and culture it at pH 6.5, 30°C, 200r / min for 24 hours as the fermentation seed liquid of Trichoderma harzianum, and store it in a refrigerator at 4°C for later use; Expanded culture: After optimization, according to the weight percentage, 1% cornmeal, 2% rice bran, 1% soybean meal, 1% sucrose, and the remainder water are used as the culture medium, and the fermentation seed liquid of Trichoderma harzianum is inoculated according to 4% of the volume of the culture medium. The culture is carried out at pH 6.5, 30°C, and 200r / min for 72h to obtain a liquid fermentation liquid, and a carrier is added at a mass volume ratio of 1:1g / mL to the liquid fermentation liquid. The carrier is zeolite powder and rice husk powder in a mass ratio of 4:6. The product is adsorbed, dried, and crushed to 80 mesh to obtain a Trichoderma harzianum fermentation product.

[0021] (2) Preparation of an anti-algae agent for controlling sea cucumber circle strong front channel algae The whole plant of the aerial part of Artemisia annua was dried at 60°C and crushed to 150 mesh, tea seed cake was crushed to 150 mesh, and the 80-mesh Trichoderma harzianum fermentation product of step (1) was used as raw materials. 0.03 mg of Artemisia annua, 0.1 mg of tea seed cake, and 0.15 mg of Trichoderma harzianum were weighed and mixed evenly to obtain a sea cucumber circle strong front channel algae inhibitor.

[0022] Example 2 (1) Purification and fermentation of Trichoderma harzianum The purification and fermentation of Trichoderma harzianum were the same as in Example 1; (2) Preparation of an anti-algae agent for controlling sea cucumber circle strong front channel algae The whole plant of the aerial part of Artemisia annua was dried at 60°C and crushed to 150 mesh, tea seed cake was crushed to 150 mesh, and the 80-mesh Trichoderma harzianum fermentation product of step (1) was used as raw materials. 0.03 mg of Artemisia annua, 0.05 mg of tea seed cake, and 0.10 mg of Trichoderma harzianum were weighed and mixed evenly to obtain a sea cucumber circle strong front channel algae inhibitor.

[0023] 1. Algae inhibition experiment (I) Purification and fermentation of Trichoderma harzianum Weigh 10 mg of commercially available Trichoderma harzianum powder, dissolve it in 10 mL of sterile distilled water, shake and mix, use an inoculation loop to streak a small amount on a PDA agar plate, and incubate at 30°C. After 1 to 2 days, colonies grow, and according to the characteristics and color of the colonies, combined with microscopic observation, pick a typical single colony (picking criteria: the single colony is flat and irregular in shape, hyphae can be seen with the naked eye, and the colony is dark green). Continue to purify twice, pick a typical colony and place it in 100 mL of PDA liquid culture medium, and culture it at pH 6.5, 30°C, 200r / min for 24 hours as the fermentation seed liquid of Trichoderma harzianum, and store it in a refrigerator at 4°C for later use; Expanded culture: After optimization, according to the weight percentage, cornmeal 1%, rice bran 2%, soybean meal 1%, sucrose 1%, according to 4% of the volume of the culture medium, inoculated with Trichoderma harzianum fermentation seed liquid to obtain liquid fermentation liquid, cultured at pH 6.5, 30°C, 200r / min for 72h, added with a carrier at a mass volume ratio of 1:1g / mL to the liquid fermentation liquid, the carrier being zeolite powder and rice husk powder in a mass ratio of 4:6, adsorbed, dried, and crushed to 80 mesh to obtain Trichoderma harzianum fermentation product.

[0024] (II) Verification of algae inhibition effect (1) The robust Amphidinium robustum was tested using f / 2 medium during the exponential growth period of Amphidinium robustum; Cultivation of robust anisopliae: f / 2 medium formula: NaNO 3 75mg / L, NaH 2 PO 4 ·H 2O 5mg / L, trace element mixture 1mL, vitamin mixture 1mL.

[0025] Preparation method: add 1 mL of each of the following four nitrogen, phosphorus, trace element mixed solutions and vitamin mixed solution stock solutions to 1 L of seawater; Nitrogen stock solution: NaNO 3 75g / L; Phosphorus stock solution: NaH 2 PO 4 ·H 2 O 5g / L; Trace element mixed solution stock solution: CuSO 4 ·5H 2 O 9.8mg / L, ZnSO 4 7H 2 O 22mg / L, CoCl 2 6H2O 10mg / L, MnCl 2 ·4H 2 O 180mg / L, NaMoO 4 ·2H 2 O 6.3mg / L, FeCl 3 6H 2 O 3.15g / L, Na 2 EDTA 4.36g / L; Vitamin mixture stock solution: Vitamin B 1100mg / L, Vitamin H 0.5mg / L, Vitamin B12 0.5mg / L.

[0026] Culture conditions: temperature (19±1)°C, illumination 4000lx, light-dark ratio: 12h:12h, to obtain a robust Amphidinium culture solution.

[0027] The algae inhibition rate calculation formula is as follows: IR=(N 0 -N S )÷N 0 ×100%; Where: IR is the inhibition rate of algae activity of the algae inhibition material (%); N 0 is the algae density of the control group (cells / mL); N S is the algae density of the experimental group.

[0028] (2) Dosage of algaecide: The experiment was conducted by adding different weights (mg) of inhibitors per ml of the culture solution of Amphidinium robustum in step (1), and the dosage was expressed as (mg / mL). The specific dosages are shown in Tables 1 to 4, 6, 7, and 9. The algae density of the culture solution of Amphidinium robustum without adding algae inhibitors was detected as the algae density of the control group; then the algae density of the culture solution of Amphidinium robustum was measured once at intervals (6h, 1d, 2d, 3d, 4d, 5d, 6d) as the algae density of the experimental group, and the algae inhibition rate of Amphidinium robustum was calculated. The results are shown in Tables 1-5, 8 and 10 (when sampling, shake it several times first, then let it stand for 30 minutes, and the sampling position is 1 cm away from the liquid surface).

[0029] ① Study on the inhibition rate of Trichoderma harzianum as an inhibitor on Algae Table 1 Algae inhibition rate of Trichoderma harzianum on robust algae (%)

[0030] ② Study on the inhibition rate of Artemisia annua as an inhibitor against Gastrodia robusta The aboveground part of Artemisia annua was dried at 60°C, crushed and passed through a 150-mesh sieve. Table 2 Algae inhibition rate of Artemisia annua against Gastrodia robusta (%)

[0031] ③ Study on the inhibition rate of tea seed cake as an inhibitor on the robust algae. The tea seed cake was crushed to 150 mesh. Table 3 Algae inhibition rate of tea seed cake on Gastrodia robusta (%)

[0032] ④Study on the inhibition rate of the combination as an inhibitor against Amphidinium robustum Artemisia annua (150 mesh), tea seed cake powder (150 mesh) and Trichoderma harzianum fermentation product (80 mesh) were prepared according to Table 4, mixed evenly, and an algaecide was obtained. Table 4 Proportions and dosages of several algaecide combinations of Artemisia annua, tea seed cake and Trichoderma harzianum combination Cumulative dose mg / mL Artemisia annua mg Tea seed cake mg Trichoderma harzianum mg 1 0.28 0.03 0.1 0.15 2 0.22 0.02 0.05 0.15 3 0.18 0.03 0.05 0.10 4 0.17 0.02 0.05 0.10 5 0.21 0.01 0.05 0.15 6 0.16 0.01 0.05 0.10 Table 5 Algae inhibition rate (%) of six different algae inhibition times combined in the above table (Table 4) combination 6h 1d 2d 3d 4d 5d 6d 1 15 63 80 99 100 100 100 2 10 35 56 64 67 64 64 3 28 70 87 95 98 98 95 4 21 28 56 61 65 59 62 5 25 41 67 70 72 74 72 6 19 38 52 58 61 63 60

[0033] From the results of algae inhibition rate in Tables 1 to 5, it can be seen that when Artemisia annua, tea seed cake and Trichoderma harzianum are used alone, the algae inhibition rate can reach more than 90% only at a large dose of 1 mg / mL, and the dosage is large. After testing, in some specific combination ratios, such as combination 1 (Example 1) and combination 3 (Example 3) in Table 4, Artemisia annua, tea seed cake and Trichoderma harzianum are used in combination with appropriate compatibility, and the algae inhibition time can reach more than 90% at low doses of 0.28 mg / mL and 0.18 mg / mL for 3 days.

[0034] The use of Artemisia annua, tea seed cake powder and Trichoderma harzianum fermented product at low concentrations of the present invention does not produce side effects on sea cucumber farming, and Artemisia annua can promote the growth of sea cucumbers and improve immunity to a certain extent, Trichoderma harzianum is a commonly used agricultural probiotic, and tea seed cake has a killing effect on fish at a certain concentration, but has no inhibitory effect on sea cucumbers. There is no side effect on sea cucumber farming at the preparation and use concentrations of Examples 1 and 2 of the present invention.

[0035] ⑤ Study on the inhibition rate of Rhizoma Coptidis, Radix Rhei, Radix Scutellariae, Radix Sophorae Flavescentis, Fructus Toosendan and Sorbus Adenophorae as inhibitors against Gastrodia robusta Table 6 Preliminary screening of the inhibitory effects of several plants and their by-products on Amphidinium robustum (%) ⑥ Study on the inhibition rate of the combination as an inhibitor against the robust algae Component A, tea seed cake powder (150 meshes) and Trichoderma harzianum fermentation product (80 meshes) were prepared according to Table 7, mixed evenly, and an algaecide was obtained. The algae inhibition experiment was carried out by using coptis root, rhubarb, scutellaria baicalensis, sophora flavescens, toosendan fruit, and arbutus glandulosa as component A, replacing the artemisia annua in Example 1 / Example 2. Table 7 Ratios and dosages of several compatible algaecides combination Cumulative dose mg / mL Component Amg Tea seed cake mg Trichoderma harzianum mg 1 0.28 0.03 0.1 0.15 2 0.18 0.03 0.05 0.10 Table 8 Algae inhibition rate (%) of different algae inhibition time of the combination in the above table (Table 7)

[0036] ⑦ Study on the algae inhibition rate of combination as an inhibitor against Amphidinium robustum Artemisia annua (150 mesh), component A and Trichoderma harzianum fermentation product (80 mesh) were prepared according to Table 9, mixed evenly, and algae inhibitors were obtained. Rhizoma Coptidis, Radix Rhei, Radix Scutellariae, Radix Sophorae Flavescentis, Fructus Toosendan and Sorbus Adenophorae were used as component A to replace the tea seed cake in Example 1 / Example 2 to carry out the algae inhibition experiment. Table 9 Ratios and dosages of several compatible algaecides combination Cumulative dose mg / mL Artemisia annua mg Component Amg Trichoderma harzianum mg 1 0.28 0.03 0.1 0.15 2 0.18 0.03 0.05 0.10 Table 10 Algae inhibition rate (%) of different algae inhibition time of the combination in the above table (Table 9) From the algae inhibition results in Tables 6 to 10, it can be seen that when Coptidis chinensis, Rhubarb, Scutellaria baicalensis, Sophora flavescens, Toosendan fruit, and Sorbus alopecuroides are used alone, at a large dose of 1 mg / mL, the algae inhibition rate can reach more than 90%, just like Artemisia annua, tea seed cake and Trichoderma harzianum. After testing, under the combination ratio of Example 1 and Example 2, Coptis chinensis, Rhubarb, Scutellaria baicalensis, Sophora flavescens, Toosendan fruit, and Sorbus alopecuroides were used to replace Artemisia annua or tea seed cake, such as Combination 1 and Combination 2 in Table 8 and Table 10. At low doses of 0.28 mg / mL and 0.18 mg / mL, the group with the best algae inhibition effect was Artemisia annua 0.03 mg, Scutellaria baicalensis 0.1 mg, and Trichoderma harzianum 0.15 mg. When the dosage was 0.28 mg / mL, the algae inhibition rate of 3d-6d was 80%-81%, and the algae inhibition rate could not reach 90%. The algae inhibition was not ideal, which was much lower than that of Example 1 and Example 2 of the present invention.

[0037] The above are only specific embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A kind of algae inhibitor for controlling sea cucumber circle strong front channel algae, characterized by: The algaecide is a mixture of artemisia annua, tea seed cake and Trichoderma harzianum.

2. The algae-inhibiting agent for controlling sea cucumber ring strength according to claim 1 is characterized in that: In terms of weight, 0.03 parts of Artemisia annua, 0.05-0.1 parts of tea seed cake, and 0.10-0.15 parts of Trichoderma harzianum are used.

3. The algae-inhibiting agent for controlling the sea cucumber circle strong front channel algae according to claim 2, characterized in that: According to the weight proportion, 0.03 parts of Artemisia annua, 0.1 parts of tea seed cake, and 0.15 parts of Trichoderma harzianum are used at a dosage of 0.28 mg / mL, and the algae inhibition rate is 99%-100% in 3-6 days.

4. The algae-inhibiting agent for controlling the sea cucumber circle strong front channel algae according to claim 2, characterized in that: According to the weight proportion, 0.03 parts of Artemisia annua, 0.05 parts of tea seed cake, and 0.10 parts of Trichoderma harzianum are used at a dosage of 0.18 mg / mL, and the algae inhibition rate is 96%-98% in 3-6 days.

5. The algae-inhibiting agent for controlling sea cucumber ring strength and anterior channel algae according to claim 1 is characterized in that: The preparation process is as follows: The whole plant of the aerial part of Artemisia annua was dried at 60°C and crushed to 150 mesh, tea seed cake powder was crushed to 150 mesh, and 80 mesh Trichoderma harzianum fermentation product was mixed evenly to obtain the sea cucumber circle strong front channel algae inhibitor.

6. The algae-inhibiting agent for controlling sea cucumber ring strength algae according to any one of claims 1 to 5, characterized in that: The preparation process of Trichoderma harzianum fermentation product is as follows: According to the percentage by weight, 1% cornmeal, 2% rice bran, 1% soybean meal, 1% sucrose, and the rest water are used as a culture medium, and 4% of the volume of the culture medium is inoculated with Trichoderma harzianum fermentation seed liquid, and cultured for 72 hours under the conditions of pH=6.5, 30°C, and 200r / min to obtain a liquid fermentation liquid; A carrier was added at a mass volume ratio of 1:1 g / mL to the liquid fermentation liquid. The carrier was zeolite powder and rice husk powder at a mass ratio of 4:

6. The mixture was adsorbed, dried, and crushed to 80 meshes to obtain a Trichoderma harzianum fermentation product.

7. Use of the algae inhibitor for controlling Amphidinium robustum as claimed in claim 1 in inhibiting Amphidinium robustum.

8. The use of the algae-inhibiting agent for controlling Amphidinium robustum in the sea cucumber circle according to claim 7 in inhibiting Amphidinium robustum, characterized in that: The algaecide is used at a concentration of 0.18-0.28 mg / mL.

9. The use of the algae-inhibiting agent for controlling Amphidinium robustum in the sea cucumber circle according to claim 8 in inhibiting Amphidinium robustum, characterized in that: The inhibition rate of the strong algae inhibitor on strong algae at a concentration of 0.18-0.28 mg / mL was 95%-99% for 72 hours.

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