An algae inhibitor for controlling sea cucumber circle strong front algae and its application

Abstract

A sea cucumber ring-encircling algae inhibitor and its application. The algae inhibitor comprises the whole aerial part of Artemisia annua, dried at 60°C and crushed to 150 mesh, and tea seed cake powder, crushed to 150 mesh and 80 mesh, and fermented Trichoderma harzianum, as raw materials. The raw materials are uniformly mixed to obtain the sea cucumber ring-encircling algae inhibitor. The sea cucumber ring-encircling algae inhibitor provided by the present invention has a simple preparation method, a wide range of raw material sources and low cost, and high algae inhibition efficiency. It can control the outbreak of Amphidinium robustum and reduce the harm of its toxin release to sea cucumbers. It is not easy to develop resistance, has no toxic side effects to sea cucumbers, and is harmless to the environment. It has significant application effects and broad market prospects.

Description

Technical Field

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

[0002] Amphidinium carterae Hulburt is a harmful red tide dinoflagellate that first appeared in the southern waters of China and has gradually spread to northern China in recent years, entering aquaculture waters. Sea cucumbers are an important aquaculture species in the Liaoning coast of northern China and are highly economically valuable. Investigations have revealed that due to the biological characteristics of sea cucumbers and their specific water quality requirements, Amphidinium carterae Hulburt is prone to outbreaks in sea cucumber aquaculture waters. In recent years, the harm of Amphidinium carterae Hulburt to sea cucumber aquaculture has become increasingly apparent. Because Amphidinium carterae Hulburt secretes toxins, outbreaks can have a significant negative impact on the growth of sea cucumbers. In severe cases, they can cause widespread mortality, resulting in significant economic losses to the sea cucumber aquaculture industry.

[0003] Currently, the primary control methods for Amphidinium robustum in sea cucumber aquaculture waters rely on herbicides and chemical disinfectants. These methods have toxic side effects on sea cucumbers, pollute the environment, and pose food safety risks. my country's sea cucumber aquaculture areas are vast, and the annual losses caused by Amphidinium robustum are substantial. Therefore, it is urgent to find a more environmentally friendly and effective algae inhibitor to control Amphidinium robustum in sea cucumber aquaculture waters. This could help control the harmful algae, protect the aquatic ecosystem, and promote the healthy development of the sea cucumber aquaculture industry. 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:

[0006] A kind of algae inhibitor for preventing and controlling sea cucumber circle strong front groove algae, its special feature is: the algae inhibitor is a mixture of artemisia annua, tea seed cake and Trichoderma harzianum.

[0007] Furthermore, in parts by 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.

[0008] Furthermore, according to the weight parts, 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 within 3 to 6 days is 99% to 100%.

[0009] Furthermore, according to the weight parts, 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 within 3-6 days is 96%-98%.

[0010] Furthermore, the preparation process is as follows:

[0011] The whole plant of the above-ground part of Artemisia annua is dried at 60° C. and crushed into 150 mesh, tea seed cake is crushed into 150 mesh, and 80 mesh Trichoderma harzianum fermentation product is mixed evenly to obtain the sea cucumber circle strong front channel algae inhibitor.

[0012] Furthermore, the preparation process of Trichoderma harzianum fermentation product is as follows:

[0013] The culture medium is composed of 1% cornmeal, 2% rice bran, 1% soybean meal, 1% sucrose, and the balance water, and the fermentation seed liquid of Trichoderma harzianum is inoculated at 4% of the volume of the culture medium. The culture is carried out at pH = 6.5, 30°C, and 200 r / min for 72 hours to obtain a liquid fermentation liquid.

[0014] 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 mesh to obtain a Trichoderma harzianum fermentation product.

[0015] An algae inhibitor for controlling the robust algae in the sea cucumber circle is used to inhibit the robust algae.

[0016] Furthermore, the algaecide is used at a concentration of 0.18-0.28 mg / mL.

[0017] Furthermore, the inhibition rate of the robust Amphidinium robustum algae inhibitor at a concentration of 0.18-0.28 mg / mL on Amphidinium robustum for 72 hours was 95%-99%.

[0018] The beneficial effects of the present invention are:

[0019] (1) The compound anti-algae effect is strong. The plant is Artemisia annua, the plant by-product is tea seed cake, and the fungus is Trichoderma harzianum. All of them 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. Tea seed cake has a killing effect on fish at a certain concentration, but has no inhibitory effect on sea cucumbers.

[0020] (2) The raw materials are widely available and low in cost. Artemisia annua, a traditional Chinese medicine for extracting artemisinin, is abundant in wild resources, which reduces its cost. Tea seed cake is a byproduct of common oil crops. It has a stable source, is inexpensive, and contains saponins that have an anti-algae effect. Trichoderma harzianum is easily available, has strong adaptability, and has a simple fermentation process, which further reduces production costs.

[0021] (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.

[0022] (4) High algae inhibition efficiency. Under the specific combination of the present invention, the algae inhibition rate can reach over 90% in 3 days at relatively low dosages (e.g., 0.28 mg / mL and 0.18 mg / mL). This highly effective algae inhibition can inhibit the outbreak of Amphidinium robustum and reduce the harm of its toxin release to sea cucumbers.

[0023] (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 safety of use.

[0024] In summary, the preparation method of the sea cucumber ring-strengthening 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 sources are wide and the cost is low. The algae inhibition efficiency is high, it is not easy to produce resistance, has no toxic side effects on sea cucumbers, and is harmless to the environment. It has significant application effects and broad market prospects. DETAILED DESCRIPTION

[0025] Example 1

[0026] (1) Purification and fermentation of Trichoderma harzianum

[0027] Weigh 10 mg of commercially available Trichoderma harzianum powder, dissolve it in 10 mL of sterile distilled water, shake and mix, use an inoculating loop to streak a small amount on a PDA agar plate, and incubate at 30°C. After 1 to 2 days of colony growth, pick a typical single colony based on the characteristics and color of the colony and observe under a microscope (picking criteria: the single colony is flat and irregular in shape, hyphae are visible to the naked eye, and the colony is dark green). Continue to purify twice, then pick a typical colony and place it in 100 mL of PDA liquid culture medium. Incubate at pH 6.5, 30°C, and 200 r / min for 24 hours as the Trichoderma harzianum fermentation seed liquid and store it in a refrigerator at 4°C for later use.

[0028] Expanded culture: After optimization, the culture medium is composed of 1% cornmeal, 2% rice bran, 1% soybean meal, 1% sucrose, and the remainder water, according to the weight percentage. The fermentation seed liquid of Trichoderma harzianum is inoculated at 4% of the volume of the culture medium. The culture is carried out at pH 6.5, 30°C, and 200 r / min for 72 hours to obtain a liquid fermentation liquid. A carrier is added at a mass volume ratio of 1:1 g / 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.

[0029] (2) Preparation of an anti-algae agent for controlling sea cucumber circle strong front channel algae

[0030] The whole plant of the aerial part of Artemisia annua was dried at 60°C and ground into 150 mesh, tea seed cake was ground into 150 mesh, and the 80 mesh fermentation product of Trichoderma harzianum in step (1) was used as raw materials. 0.03 mg of Artemisia annua and 0.1 mg of tea seed cake were weighed and 0.15 mg of Trichoderma harzianum were mixed uniformly to obtain the sea cucumber circle strong front channel algae inhibitor.

[0031] Example 2

[0032] (1) Purification and fermentation of Trichoderma harzianum

[0033] The purification and fermentation of Trichoderma harzianum were the same as in Example 1;

[0034] (2) Preparation of an anti-algae agent for controlling sea cucumber circle strong front channel algae

[0035] The whole plant of the aerial part of Artemisia annua was dried at 60°C and ground into 150 mesh, tea seed cake was ground into 150 mesh, and the 80 mesh fermentation product of Trichoderma harzianum in step (1) was used as raw materials. 0.03 mg of the raw material Artemisia annua, 0.05 mg of tea seed cake, and 0.10 mg of Trichoderma harzianum were weighed and mixed evenly to obtain the sea cucumber circle strong front channel algae inhibitor.

[0036] 1. Algae inhibition experiment (I) Purification and fermentation of Trichoderma harzianum

[0037] Weigh 10 mg of commercially available Trichoderma harzianum powder, dissolve it in 10 mL of sterile distilled water, shake and mix, use an inoculating loop to streak a small amount on a PDA agar plate, and incubate at 30°C. After 1 to 2 days of colony growth, pick a typical single colony based on the characteristics and color of the colony and observe under a microscope (picking criteria: the single colony is flat and irregular in shape, hyphae are visible to the naked eye, and the colony is dark green). Continue to purify twice, then pick a typical colony and place it in 100 mL of PDA liquid culture medium. Incubate at pH 6.5, 30°C, and 200 r / min for 24 hours as the Trichoderma harzianum fermentation seed liquid and store it in a refrigerator at 4°C for later use.

[0038] Expanded culture: After optimization, according to the weight percentage, cornmeal 1%, rice bran 2%, soybean meal 1%, and sucrose 1% were inoculated with Trichoderma harzianum fermentation seed liquid at 4% of the culture medium volume to obtain liquid fermentation liquid, and cultured at pH 6.5, 30°C, and 200 r / min for 72 hours. 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 in a mass ratio of 4:6. The product was adsorbed, dried, and crushed to 80 mesh to obtain Trichoderma harzianum fermentation product.

[0039] (2) Verification of algae inhibition effect

[0040] (1) The robust algae were grown in f / 2 culture medium during the exponential growth period of the robust algae.

[0041] Culture of Amphidinium robustum:

[0042] f / 2 culture medium formula: NaNO3 75 mg / L, NaH2PO4·H2O 5 mg / L, trace element mixture 1 mL, vitamin mixture 1 mL.

[0043] Preparation method: add 1 mL each of the following four nitrogen, phosphorus, trace element mixtures and vitamin mixture stock solutions to 1 L of seawater;

[0044] Nitrogen stock solution: NaNO3 75g / L;

[0045] Phosphorus stock solution: NaH2PO4·H2O 5g / L;

[0046] Trace element mixture stock solution: CuSO4·5H2O 9.8mg / L, ZnSO4·7H2O 22mg / L, CoCl2·6H2O 10mg / L, MnCl2·4H2O 180mg / L, NaMoO4·2H2O 6.3mg / L, FeCl3·6H2O 3.15g / L, Na2EDTA 4.36g / L; vitamin mixture stock solution: vitamin B 1100mg / L, vitamin H 0.5mg / L, vitamin B12 0.5mg / L.

[0047] Culture conditions: temperature (19±1)°C, illumination 4000 lx, light-dark ratio: 12h:12h, to obtain a robust Amphidinium culture solution.

[0048] The algae inhibition rate calculation formula is as follows:

[0049] IR=(N0-N S )÷N0×100%;

[0050] Where: IR is the inhibition rate of algae activity by the algae inhibitory material (%); N0 is the algae density of the control group (cells / mL); N S is the algae density of the experimental group.

[0051] (2) Dosage of algaecide:

[0052] The experiment was conducted by adding different weights (mg) of inhibitors per milliliter of the culture medium 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.

[0053] The algal density of the culture solution of Amphidinium robustum without adding algaecide was detected as the algal density of the control group; then the algal density of the culture solution of Amphidinium robustum was measured once at intervals of 6h, 1d, 2d, 3d, 4d, 5d, 6d as the algal density of the experimental group, and the algal 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 30min, and the sampling position is 1cm away from the liquid surface).

[0054] ① Study on the inhibition rate of Trichoderma harzianum as an inhibitor against Gastrodia robusta

[0055] Table 1 Algae inhibition rate of Trichoderma harzianum against Gastrodia robusta (%)

[0056]

[0057] ② Study on the inhibition rate of Artemisia annua as an inhibitor against Gymnodiella robusta The aerial parts of Artemisia annua were dried at 60℃, crushed and passed through a 150-mesh sieve.

[0058] Table 2 Algae inhibition rate of Artemisia annua against Gastrodia robusta (%)

[0059]

[0060] 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.

[0061] Table 3 Algal inhibition rate of tea seed cake on Gastrodia robusta (%)

[0062]

[0063]

[0064] ④Study on the inhibition rate of the combination as an inhibitor against Amphidinium robustum

[0065] 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.

[0066] Table 4 Ratios and dosages of several compatible algaecides of Artemisia annua, tea seed cake and Trichoderma harzianum

[0067] 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

[0068] Table 5 Algae inhibition rate (%) of six different algae inhibition times combined in the above table (Table 4)

[0069] 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

[0070] As shown in Tables 1-5, the algae inhibition rates for Artemisia annua, tea seed cake, and Trichoderma harzianum, when used alone, only achieved an algae inhibition rate exceeding 90% at a high dose of 1 mg / mL, a high dosage being required. Testing also revealed that, in certain specific combinations, such as Combination 1 (Example 1) and Combination 3 (Example 3) in Table 4, the combined use of Artemisia annua, tea seed cake, and Trichoderma harzianum achieved an algae inhibition rate exceeding 90% within 3 days at low doses of 0.28 mg / mL and 0.18 mg / mL, respectively.

[0071] The use of Artemisia annua, tea seed cake powder, and Trichoderma harzianum fermentation products at low concentrations in the present invention does not produce any side effects on sea cucumber farming. 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. Tea seed cake has a killing effect on fish at a certain concentration but has no inhibitory effect on sea cucumbers. There are no side effects on sea cucumber farming at the preparations and concentrations used in Examples 1 and 2 of the present invention.

[0072] ⑤ Study on the inhibition rate of Rhizoma Coptidis, Rheum officinale, Scutellaria baicalensis, Sophora flavescens, Toosendan fruit and Aralia oleracea as inhibitors against Gastrodia robusta

[0073] Table 6 Preliminary screening of the inhibitory effects of several plants and their by-products on Amphidinium robustum (%)

[0074]

[0075] ⑥Study on the inhibition rate of the combination as an inhibitor against Amphidinium robustum

[0076] Component A, tea seed cake powder (150 mesh) and Trichoderma harzianum fermentation product (80 mesh) were prepared according to Table 7, mixed evenly, and an algaecide was obtained.

[0077] The algae inhibition experiment was carried out using Rhizoma Coptidis, Rhei Radix, Scutellariae Baicalensis, Sophora flavescens, Toosendan Fruit, and Aronia oleracea as component A, replacing the Artemisia annua in Example 1 / Example 2.

[0078] Table 7 Ratios and dosages of several compatible algaecides

[0079] 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

[0080] Table 8 Algae inhibition rate (%) of different algae inhibition times of the combination in the above table (Table 7)

[0081]

[0082]

[0083] ⑦ Study on the inhibition rate of combination as an inhibitor against Amphidinium robustum Artemisia annua (150 mesh), component A and Trichoderma harzianum fermentation product (80 mesh) were respectively prepared according to Table 9, mixed evenly, and obtained an algaecide.

[0084] The algae inhibition experiment was carried out by using Rhizoma Coptidis, Rhei Radix, Scutellariae Baicalensis, Sophora flavescens, Toosendan Fruit, and Aronia oleracea as component A, replacing the tea seed cake in Example 1 / Example 2.

[0085] Table 9 Ratios and dosages of several compatible algaecides

[0086] 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

[0087] Table 10 Algae inhibition rate (%) of different algae inhibition times of the combination in the above table (Table 9)

[0088]

[0089]

[0090] From the algae inhibition rate results in Tables 6 to 10, it can be seen that when Coptidis rhizome, Rhubarb, Scutellaria baicalensis, Sophora flavescens, Toosendan fruit, and Aralia nigra 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, at the combination ratios of Example 1 and Example 2, Coptis chinensis, Rhubarb, Scutellaria baicalensis, Sophora flavescens, Toosendan fruit, and Arbutus glandulosa were used to replace Artemisia annua or tea seed cake, such as Combination 1 and Combination 2 in Tables 8 and 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 for 3d-6d was 80%-81%, and the algae inhibition rate could not reach 90%. The algae inhibition was not ideal, which was far lower than that of Example 1 and Example 2 of the present invention.

[0091] The above are merely specific embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A kind of algae inhibitor for controlling sea cucumber circle strong front channel algae, characterized by: The algae inhibitor is a mixture of Artemisia annua, tea seed cake and Trichoderma harzianum fermentation product; In terms of weight, the ingredients include 0.03 parts of Artemisia annua, 0.05-0.1 parts of tea seed cake, and 0.10-0.15 parts of Trichoderma harzianum fermentation product.

2. The algae inhibitor for controlling the sea cucumber circle strong front algae according to claim 1, characterized in that: According to the weight ratio, 0.03 parts of Artemisia annua, 0.1 parts of tea seed cake, and 0.15 parts of Trichoderma harzianum fermentation product are used at a dosage of 0.28 mg / mL, and the algae inhibition rate within 3-6 days is 99%-100%.

3. The algae-inhibiting agent for controlling the sea cucumber circle strong front algae according to claim 1, characterized in that: According to the weight ratio, 0.03 parts of Artemisia annua, 0.05 parts of tea seed cake, and 0.10 parts of Trichoderma harzianum fermentation product are used at a dosage of 0.18 mg / mL, and the algae inhibition rate within 3-6 days is 96%-98%.

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

5. The algae inhibitor for controlling the sea cucumber circle strong front algae according to any one of claims 1 to 4, characterized in that: The preparation process of Trichoderma harzianum fermentation product is as follows: The culture medium was prepared by inoculating 4% of the volume of the culture medium with 1% cornmeal, 2% rice bran, 1% soybean meal, 1% sucrose, and the balance water, and incubating the culture medium at pH 6.5, 30°C, and 200 rpm for 72 hours 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 mesh to obtain a Trichoderma harzianum fermentation product.

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

7. The application of the algae inhibitor for controlling Amphidinium robustum in the sea cucumber circle according to claim 6 in inhibiting Amphidinium robustum is characterized in that: The algaecide is used at a concentration of 0.18-0.28 mg / mL.

8. The application of the algae inhibitor for controlling Amphidinium robustum in the sea cucumber circle according to claim 7 in inhibiting Amphidinium robustum is characterized in that: The inhibition rate of the strong algae inhibitor at a concentration of 0.18-0.28 mg / mL on strong algae for 72 hours is 95%-99%.

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