Creation and application of multi-element microorganism wettable powder for treating rice diseases and insect pests

Abstract

The invention provides preparation of trichoderma-spore-metarhizium anisopliae multi-element wettable powder and an application method of the trichoderma-spore-metarhizium anisopliae multi-element wettable powder in prevention and treatment of rice diseases and insect pests. Wherein the trichoderma, the bacillus and the metarhizium anisopliae can grow in an affinity manner on a PDA (Personal Digital Assistant) flat plate, and the form diagram of the wettable powder is shown as an attached drawing 1 in the abstract of the specification; the number of trichoderma viable spores in the wettable powder is 2.96 * 10 < 8 > CFU / g, the amount of spore viable bacteria is 3.58 * 10 < 8 > CFU / g, and the amount of green muscardine silkworm is 1.42 * 10 < 9 > CFU / g. After the paddy rice sprayed with the wettable powder is applied for 28 days, compared with clear water treatment, the plant has the advantages that the root length is increased by 100%, the plant height is increased by 83%, and the fresh weight is 5 times that of clear water control. Under the condition that the wettable powder is sprayed on the rice for 14 days, the control effects of the rice sheath blight disease, the rice blast and the bacterial leaf blight are 65.51%, 68.75% and 61.90% respectively. The indoor toxicity of the wettable powder on the rice leaf folder is measured, and the corrected death rate of the wettable powder is 89%. The invention provides possibility for prevention and control and growth promotion of rice diseases and insect pests, and has important significance for effective prevention and control of rice diseases and insect pests.

Description

1. Technical Field

[0001] The present invention relates to the preparation and application of a drug in the technical field of agricultural pest control, in particular to a preparation method and application of a Trichoderma-spore-Metarhizium wettable powder for controlling rice pests. 2. Background Technology

[0002] Rice is the world's most important food crop, yet rice pests and diseases have a significant impact on rice yield and quality. Rice sheath blight, rice blast, and bacterial leaf blight are all common rice diseases that, in severe cases, can kill the entire plant. Currently, only a few rice varieties are resistant to these diseases, and even these varieties are less resistant to multiple diseases. Rice borers (such as the striped stem borer, yellow stem borer, and rice leaf roller) are also common on rice.

[0003] Measures for controlling rice pests and diseases primarily include the breeding and utilization of disease-resistant varieties, pesticide control, cultivation management, and biological control. However, chemical control currently predominates in the prevention and control of these pests and diseases, but this can easily lead to pesticide resistance, damage the ecological environment, and create pesticide residues. Driven by the need for green, safe, and sustainable agricultural development, a growing trend is toward biological control to address rice pest and disease issues. However, biological control often relies on a single biocontrol agent to control a single disease, resulting in low control efficiency and inconsistent results.

[0004] Therefore, developing a biological agent that can simultaneously control multiple pests and diseases is particularly important. Trichoderma effectively inhibits the growth of various plant pathogens, Bacillus promotes plant growth and enhances plant disease resistance, and Metarhizium significantly controls a wide range of insect pests. This combination can not only reduce the use of chemical pesticides and environmental pollution, but also improve rice yield and quality, achieving sustainable agricultural production. 3. Summary of the Invention

[0005] One object of the present invention is to control rice sheath blight, rice blast, bacterial leaf blight and rice leaf roller.

[0006] An object of the present invention is to prepare and use a wettable powder of a composite bacterial agent, which is composed of a Trichoderma sp. HN-034, a Bacillus sp. B-HN-1 and a Metarhizium anisopliae Ma-HN-1; the function of the composite bacterial agent is to inhibit the occurrence of rice sheath blight, rice blast, bacterial leaf blight and rice leaf roller.

[0007] The technical solution adopted by the present invention is: a Trichoderma sp. HN-034, the preservation number of the strain is CGMCC No.40945.

[0008] A Bacillus sp. B-HN-1 strain, the deposit number of which is CGMCC No.29276.

[0009] The invention relates to a strain of Metarhizium anisopliae Ma-HN-1, the preservation number of which is CGMCC No.40949.

[0010] The above-mentioned Trichoderma sp. HN-034 is used to inhibit the growth of plant pathogens.

[0011] The above-mentioned Bacillus sp. B-HN-1 is used to inhibit the growth of plant pathogens.

[0012] The Metarhizium anisopliae Ma-HN-1 is used to parasitize and kill rice leaf rollers.

[0013] In the above scheme, the pathogens are Rhizoctonia solani, Pyricularia oryzae, and Xanthomonas oryzae.

[0014] The viable spore count of Trichoderma in the wettable powder in the above scheme is 2.96×10 8 CFU / g, the amount of viable spore bacteria is 3.58×10 8 CFU / g, green muscardine is 1.42×10 9 CFU / g, the root length, plant height and fresh weight of the WP treatment group were 21.67cm, 42.57cm and 1.63g, respectively, which had a significant growth-promoting effect.

[0015] The control efficacy of the wettable powder WP in the above-mentioned file against rice sheath blight, rice blast and bacterial leaf blight is 65.51%, 68.75% and 61.90% respectively.

[0016] The corrected mortality of the wettable powder WP against the rice leaf folder in the above scheme was 89%.

[0017] Beneficial effects:

[0018] The multi-microorganism wettable powder provided by the present invention has good effects of promoting the growth of rice plants and preventing and controlling rice diseases and rice leaf rollers.

[0019] Preservation Instructions

[0020] Strain name: Trichoderma HN-034;

[0021] Latin name: Trichoderma sp.;

[0022] Deposit number: CGMCC No.40945;

[0023] Depository: China General Microbiology Center (CGMCC);

[0024] Address: No. 1, Beichen West Road, Chaoyang District, Beijing;

[0025] Date of preservation: December 11, 2023.

[0026] Strain name: Bacillus B-HN-1;

[0027] Latin name: Bacillus sp.

[0028] Deposit number: CGMCC No.29726;

[0029] Depository: China General Microbiology Center (CGMCC);

[0030] Address: No. 1, Beichen West Road, Chaoyang District, Beijing;

[0031] Date of preservation: December 11, 2023.

[0032] Strain name: Metarhizium anisopliae Ma-HN-1;

[0033] Latin name: Metarhizium anisopliae;

[0034] Deposit number: CGMCC No.40949;

[0035] Depository: China General Microbiology Center (CGMCC);

[0036] Address: No. 1, Beichen West Road, Chaoyang District, Beijing;

[0037] Date of preservation: December 11, 2023. IV. Description of the Figures

[0038] Figure 1 Compatibility Identification of Trichoderma-Spore-Metarhizium on Plate

[0039] Figure 2 The form of Trichoderma-Spore-Metarhizium multi-microbial wettable powder

[0040] Figure 3 Effect of Trichoderma-Spore-Metarhizium Multi-microbial Wettable Powder on Rice Growth Promotion

[0041] Figure 4 Control effect of Trichoderma-Bacillus-Metarhizium multi-microbial wettable powder on rice sheath blight

[0042] Figure 5 Effect of Trichoderma-Bacillus-Metarhizium multi-microbial wettable powder on rice blast

[0043] Figure 6 The efficacy of Trichoderma-Spore-Metarhizium multi-microbial wettable powder against bacterial blight

[0044] Figure 7 Effect of Trichoderma-Bacillus-Metarhizium multi-microbial wettable powder on rice leaf roller V. Specific Implementation Methods

[0045] The present invention will be described in detail below with reference to specific embodiments, which are intended to help understand but not limit the present invention.

[0046] Example 1: Affinity test of Trichoderma, spores and Metarhizium

[0047] Draw a line about 20 mm away from the edge of the plate to inoculate Bacillus, take a 5 mm diameter cake of Trichoderma and Metarhizium anisopliae at the edge of the colony, and inoculate the cakes at 50 mm intervals in the center of the marked area to observe whether the three bacteria can grow normally on the same plate and whether an inhibition zone is produced.

[0048] After inoculation, place the culture dish in a constant temperature incubator at 25–28°C and observe the growth dynamics of Trichoderma and Bacillus at regular intervals every day. If no obvious inhibition zone is formed between the two and they can contact each other, it indicates that there is good affinity between Trichoderma and Bacillus. The plate affinity images of Trichoderma HN-034, Bacillus velezensis B-HN-1 and Metarhizium anisopliae Ma-HN-1 are shown in the attached figure. Figure 1 shown.

[0049] Example 2: Preparation method of Trichoderma-spore powder and Metarhizium anisopliae powder

[0050] 1. Activation treatment of the required strain.

[0051] Trichoderma with the preservation number of CGMCC No.40945 and Metarhizium anisopliae with the preservation number of CGMCC No.40949 were inoculated on PDA plate culture medium respectively and cultured at a constant temperature of 28°C for 72 hours; Bacillus with the preservation number of CGMCC NO.29276 was inoculated on solid LB plate culture medium and cultured at a constant temperature of 37°C for 72 hours.

[0052] PDA medium: 200 g potatoes, 20 g glucose, 18 g agar powder, 1 L distilled water.

[0053] LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 18 g agar powder, and 1 L distilled water.

[0054] 2. Preparation of spore suspension.

[0055] Preparation of Trichoderma spore suspension: Inoculate the activated Trichoderma HN-034 strain on a PDA plate and culture at 28°C for 5-7 days. Use a spreader to scrape the spores on the PDA plate and prepare a spore suspension with a concentration of 10 6 CFU / L, stored at 4℃ until use.

[0056] Preparation of Metarhizium anisopliae spore suspension: Inoculate the activated Metarhizium anisopliae Ma-HN-1 strain on a PDA plate and culture at 28°C for 5-7 days. Use a spreader to scrape the spores on the PDA plate and prepare a spore suspension with a concentration of 10 6 CFU / L, stored at 4℃ until use.

[0057] Preparation of bacterial culture: Pick activated bacterial colonies and inoculate them into liquid LB medium respectively, culture them at 37℃ and 180r / min for 72h, and the concentration is about 10 8 CFU / L, stored at 4℃ until use.

[0058] LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 1 L distilled water.

[0059] The optimal fermentation conditions for the Trichoderma-Bacillus co-fermentation of the present invention are: 8g of rice husk, 8g of corn flour, 4g of wheat bran, 40% water content, and 10g / L of glucose per 20g of culture medium. After 7 days of co-fermentation under these conditions, the fermentation product was dried, crushed, and passed through a 200-mesh sieve. The content of the obtained Trichoderma spore powder was 7.51×10 9 CFU / g, the Bacillus cell yield was 9.22×10 9 CFU / g.

[0060] In addition, the present invention also provides a method for solid fermentation of Metarhizium anisopliae, using ordinary rice as a culture medium, with a water content of 40% per 20 g of rice. After 7 days, the rice is dried, crushed and passed through a 200 mesh sieve, and the content of Metarhizium anisopliae spore powder obtained is 2.68×10 9 CFU / g.

[0061] Example 3: A method for preparing Trichoderma-Spore-Metarhizium WP

[0062] The powder of Trichoderma-Bacillus and the powder of Metarhizium anisopliae were mixed in a ratio of 1:9; the content of the mixed powder was 20%, the content of Tween-20 was 5%, the content of sodium carboxymethyl cellulose was 8%, the content of lecithin was 0.4%, and the content of white carbon black was 66.6%. After grinding, mixing, drying and other steps, the Trichoderma-spore-Metarhizium anisopliae WP was prepared, in which the number of viable Trichoderma spores was 2.96×10 8 CFU / g, the amount of viable spore bacteria is 3.58×10 8 CFU / g, green muscardine is 1.42×10 9 CFU / g, its morphology is attached Figure 2 .

[0063] Example 4: Effect of Trichoderma-Spore-Metarhizium WP on Rice Growth Promotion

[0064] Take 1g of WP and dilute it to 100mL with sterile water for later use.

[0065] Rice seeds with uniform germination were sown into plastic pots of 25 cm in height, 16 cm in length and 16 cm in width filled with sterile soil, and cultured in a greenhouse at a temperature of 30° C. and a relative humidity of 90%.

[0066] The experimental groups were divided into 1 control group and 4 experimental groups, with 3 rice seedlings in each group: the control group was inoculated with sterile water; the experimental groups were inoculated with 20 mL of WP dilution; 20 mL of 1×10 7 CFU / mL HN-034 spore suspension and 20mL 1×10 7 CFU / mL Ma-HN-1 spore suspension; 20mL 1×10 7 CFU / mL ZJ bacterial suspension, observe and measure plant height and root length after 28 days, and repeat 3 times.

[0067] Table 1 Ability of different treatments of the present invention to promote rice plant growth

[0068]

[0069] It can be seen that the wettable powder WP of the present invention is better than the use of Trichoderma HN-034 and Bacillus B-HN-1 alone in promoting plant growth. Figure 3 shown.

[0070] Example 4: Effect of Trichoderma-Spore-Metarhizium WP on Rice Disease Control

[0071] Weigh 1g of wettable powder (WP) and dissolve it in 100mL of sterile water to prepare a 0.01g / mL solution. Sow evenly germinated rice seeds in sterile plastic pots (25×16×16cm) with soil and incubate in a greenhouse at 30°C and 90% humidity until the tillering stage. Different inoculation treatments were applied depending on the disease type:

[0072] (1) Sheath blight: 6 mm cakes of bacteria were prepared from the strain cultured on PDA for 2 days and inoculated onto the base of the plant and leaves using insect pins;

[0073] (2) Rice blast: In addition to the inoculation of the fungus cake, spray 10 6 -10 7 CFU / mL spore suspension on leaf surface

[0074] (3) Bacterial leaf blight: Use an inoculation needle to scratch the leaves and then spray the bacterial solution.

[0075] Two days after inoculation, treatments were applied: the sheath blight group was sprayed with a Trichoderma-spore-Metarhizium preparation, a 300-fold solution of 4% Jinggangmycin, and water; the rice blast and bacterial blight groups were sprayed with a 300-fold solution of 6% Kasugamycin. All treatments were replicated three times, with three plants per replicate. Water was used as a control, and the leaves were sprayed until water dripped. For the bacterial blight group, the inoculation site was kept moist. Fourteen days after treatment, disease severity was assessed according to national standards (GB / T 17980.20-2000, GB / T 15790-2009): sheath blight was graded based on the location of lesions on the leaf sheath (0-9), blast was graded based on the number and area of ​​lesions (0-9), and bacterial blight was graded based on the percentage of lesion area (0-9). Disease severity was calculated using a disease index = Σ(number of grades × corresponding number of plants) / (total number of plants × 9) × 100. The control effect was calculated as follows: for sheath blight, [1-(disease index before control drug × disease index after treatment drug) / (disease index after control drug × disease index before treatment drug)]×100; for other diseases, [(control disease index-treatment disease index) / control disease index]×100; for bacterial blight, the lesion inhibition rate was additionally calculated.

[0076] Table 2 The control effect of Trichoderma-Spore-Metarhizium WP on rice diseases

[0077]

[0078] It can be seen that the effect of the wettable powder WP of the present invention in preventing and controlling rice diseases is not much different from that of pesticides. Figure 4 、 5 , as shown in 6.

[0079] Example 5: Indoor toxicity determination of Trichoderma-spore-Metarhizium anisopliae WP against rice leaf folder

[0080] Weigh 1g of wettable powder (WP) and dissolve it in 100mL of sterile water to prepare a 0.01g / mL solution for later use. Use a brush tool to gently pick up the third-instar larvae from the head backwards, then place it in a solution of known concentration for 30s of infection, pick out the larvae, place them in a prepared culture dish, and add rice leaves to the culture dish. Cover the lid and bottom of the culture dish with a layer of black cloth to prevent the larvae from escaping through the gaps. Keep them in an incubator at 26℃ and a relative humidity of 80%. Each experimental group had 40 larvae, and the experiment was repeated 4 times. The control group used a sterile aqueous solution containing 0.5% Tween-80 and 6ppm of avermectin. The death of insects in each experimental group was observed and recorded regularly every day, and the observation was continued for 7 days, and the mortality rate was calculated.

[0081] Table 3 Indoor toxicity test of Trichoderma-spore-Metarhizium anisopliae WP against rice leaf folder

[0082]

[0083] It can be seen that the effect of the wettable powder WP of the present invention in controlling rice leaf roller is not much different from that of pesticides. The parasitic effect of WP is shown in the attached figure. Figure 7 Shown in Sequence Table 1:

[0084]

[0085]

[0086] Sequence Listing 2:

[0087]

[0088]

[0089] Sequence Listing 3:

[0090]

[0091]

Claims

1. A polymicrobial wettable powder, characterized in that The product contains Trichoderma longibrachiatum HN-034, Bacillus B-HN-1 and Metarhizium anisopliae Ma-HN-1, and the number of viable spores of Trichoderma is 2.96×10 8 CFU / g, the amount of viable spore bacteria is 3.58×10 8 CFU / g, green muscardine is 1.42×10 9 CFU / g.

2. The polymicrobial wettable powder according to claim 1, characterized in that The mixing ratio of the Trichoderma HN-034-Bacillus B-HN-1 fungus powder and the Metarhizium anisopliae Ma-HN-1 fungus powder is 1:

9.

3. The polymicrobial wettable powder according to claim 1, characterized in that The polymicrobial wettable powder further comprises Tween-20, sodium carboxymethyl cellulose, lecithin and white carbon black.

4. The polymicrobial wettable powder according to claim 3, characterized in that The content of Tween-20 is 5%, the content of sodium carboxymethyl cellulose is 8%, the content of lecithin is 0.4%, and the content of white carbon black is 66.6%.

5. The polymicrobial wettable powder according to claim 1, characterized in that The multi-microorganism wettable powder has a prevention effect of 65.51%, 68.75% and 61.90% on rice sheath blight, rice blast and bacterial leaf blight respectively.

6. The polymicrobial wettable powder according to claim 1, characterized in that The corrected mortality rate of the multi-microbial wettable powder to rice leaf folder is 89%.

7. The polymicrobial wettable powder according to claim 1, characterized in that The multi-microorganism wettable powder has the effects of promoting the growth of rice plants and preventing and controlling rice diseases and rice leaf rollers.

8. The polymicrobial wettable powder according to claim 1, characterized in that The Latin name of the longibrachiatum Trichoderma longibrachiatum HN-034 is CGMCC No. 40945, the preservation institution is the General Microbiology Center of the China Culture Collection Administration (CGMCC), and the preservation date is December 11, 2023.

9. The polymicrobial wettable powder according to claim 1, characterized in that The Latin name of the Bacillus B-HN-1 is Bacillus sp., the preservation number is CGMCC No. 29726, the preservation institution is the General Microbiology Center of the China Culture Collection Administration (CGMCC), and the preservation date is December 11, 2023.

10. The polymicrobial wettable powder according to claim 1, characterized in that The Latin name of the Metarhizium anisopliae Ma-HN-1 is Metarhizium anisopliae, the preservation number is CGMCC No.40949, the preservation institution is the General Microbiology Center of the China Culture Collection Administration (CGMCC), and the preservation date is December 11, 2023.

11. The method for preparing the polymicrobial wettable powder according to claim 1, comprising the following steps: a) inoculating Trichoderma longibrachiatum HN-034, Bacillus sp. B-HN-1, and Metarhizium anisopliae Ma-HN-1 on appropriate culture media for activation culture; b) preparing a solid co-fermentation product of Trichoderma longibrachiatum HN-034-Bacillus B-HN-1 and a solid fermentation product of Metarhizium anisopliae Ma-HN-1; c) mixing the fungus powders of Trichoderma longibrachiatum HN-034-Bacillus B-HN-1 and Metarhizium anisopliae Ma-HN-1 in a certain proportion; d) drying the mixed bacterial powder, crushing it and passing it through a 200-mesh sieve, and compounding it with the adjuvant according to claim 4 to obtain the polymicrobial wettable powder.

12. The preparation method according to claim 11, characterized in that The optimal co-fermentation conditions of Trichoderma HN-034 and Bacillus B-HN-1 are: 8g rice husk, 8g corn flour, 4g wheat bran, 40% water content, and 10g / L glucose per 20g culture medium.

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