Microbial seed treatment agent for preventing and treating bakanae disease of rice and application of microbial seed treatment agent

Abstract

The invention relates to a microbial seed treatment agent and application thereof in agricultural production, and belongs to the field of research and development of agricultural microbial products. The bacillus Bs916 has a relatively strong effect of inhibiting pathogenic bacteria of the rice bakanae disease. The invention provides a seed treatment agent which is prepared by taking Bs916 living spores as active ingredients and mixing auxiliaries, the seed treatment agent is red suspension liquid, the spore content is more than or equal to 3 * 10 < 9 > CFU / mL, the suspension rate is more than 80%, and the pH is 6.0-7.0. The bacillus Bs916 seed treating agent has the effects of promoting rice seed germination and seedling growth and preventing and treating rice bakanae disease. When the seed treatment agent is respectively mixed with tebuconazole (250mg / L), validamycin (50mg / L) and agricultural resistance 120 (200mg / L), the effects of preventing and treating the rice bakanae disease are respectively 87.54 percent, 86.74 percent and 88.05 percent. The invention discloses a microbial seed treatment agent capable of promoting growth and preventing and treating rice bakanae disease, which can stabilize the prevention effect, reduce the dosage of chemical pesticide and reduce pesticide residue when being mixed with chemical pesticide, and has potential application value and market development prospect in agricultural production.

Description

Technical Field

[0001] The present invention relates to a biological seed treatment agent whose active ingredients are living microorganisms and its application in agricultural production, belonging to the field of agricultural microbial products and technology research and development. Background Art

[0002] Rice Bakanae Disease, also known as leggy growth disease and white stalk disease, occurs in all rice-growing areas of my country. It can generally cause a yield reduction of 10% to 20%, and in severe cases, more than 50% or even complete crop failure, severely impacting yield. Furthermore, it produces various toxins, including fumonisins, that affect rice quality. Rice Bakanae Disease is a systemic seed-borne disease caused by the Gibberella fujikuroi species complex (GFSC). The GFSC species complex primarily includes Fusarium fujikuroi, Fusarium proliferatum, and Fusarium verticillioides, with Fusarium fujikuroi being the primary pathogen. Infected seeds are the primary source of infection for rice Bakanae Disease. Therefore, using disease-free seeds is key to controlling the occurrence of the disease. Treating seeds with seed treatment agents is one of the economical and effective methods to prevent and control soil-borne, seed-borne diseases and underground pests.

[0003] Treating rice seeds with chemical agents for seed soaking or coating is currently a key measure for controlling bakanae disease. Commonly used chemical agents include metalaxyl-M, fludioxonil, prochloraz, cyproconazole, tebuconazole, ethionyl, carbendazim, and thiram. In addition to single-agent applications, their effectiveness can be further enhanced through combination therapy, such as prochloraz-M combined with carbendazim, metalaxyl-M combined with fludioxonil, and tebuconazole combined with fludioxonil. With the long-term use of chemical agents, resistance to rice bakanae disease has gradually increased, increasing drug usage and resulting in pesticide residues, which harm the environment and human and animal health. Therefore, replacing the active ingredients of traditional chemical agents with environmentally friendly, animal-safe microorganisms or their secondary metabolites is becoming a key area of ​​product research and development. With the in-depth study of rhizosphere microbiome theory, the research, industrialization, and application of microorganisms in plant protection and plant nutrition have become a potential market growth area.

[0004] The China Pesticide Information Network shows that the biological pesticide ingredients currently developed in my country mainly include Bacillus, Pseudomonas, Trichoderma, Streptomyces and Rhizobia, as well as microbial secondary metabolites (antibiotics). Although there are fewer registered biological seed treatment agents, such as Yangzhou Lvyuan Biotechnology Co., Ltd. registered Bacillus subtilis suspension seed coating agent and Wuhan Kono Technology Co., Ltd. registered Bacillus polymyxa seed treatment agent, biological seed treatment agents have become a hot spot for research and development in the industry. The China Invention Patent Database shows that invention patent CN201611096156.X discloses the use of three beneficial microorganisms, Bacillus polymyxa, Bacillus cereus and Bacillus pumilus, to prepare biological seed coating agents to promote the germination of seeds of crops such as rice and corn, and prevent and control underground pests, rice seedling disease, corn smut and stem base rot. Invention patent CN202211440330.3 discloses a biological seed coating agent prepared using Bacillus polymyxa and Bacillus amyloliquefaciens with iron-containing compounds to improve seed germination rate, enhance seedling quality, and control wheat sheath blight, soybean root rot, and alfalfa damping-off. Invention patent CN201310722789.7 discloses a seed treatment agent containing Bacillus subtilis, its preparation method, and its efficacy in controlling crop diseases such as wilt, sheath blight, take-all disease, rice blast, gray mold, powdery mildew, and bacterial wilt. Biological seed treatment agents offer the following advantages over chemical seed treatments: 1. Environmentally friendly, harmless to humans and animals, and safe for seeds; 2. Easy to decompose in the environment, leaving no residue or pollution; 3. Improve soil structure and reshape the microhabitat for beneficial microorganisms in the rhizosphere. However, microbial pesticides have poor stability in field control, and exploring diverse usage models is needed to improve their stability and effectiveness. There is an urgent production and market demand for research on high-quality, safe biological seed treatment agents and their efficient and stable use methods.

[0005] The Rice Disease Biological Control Research Team of the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences has been engaged in the exploration, storage, research and development, demonstration and promotion of plant disease biocontrol resources for nearly 30 years. They have developed biological pesticides such as "Wenquning" and "Yebanning" for the prevention and control of rice sheath blight and bacterial leaf streak. The main active ingredient of the product "Wenquning" is Bacillus subtilis Bs916, which was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration at the Institute of Microbiology, Chinese Academy of Sciences on September 30, 2002, with the deposit number CGMCC No. 0808. The inventor of this patent determined through a plate confrontation experiment that Bs916 has a strong inhibitory effect on various pathogenic Fusarium spp. of rice seedling disease, and successfully prepared a microbial seed treatment agent using the live spore fermentation liquid of Bs916 as the main active ingredient and mixed it with relevant pesticide adjuvants. The agent is a red viscous suspension with a live bacteria (spore) content of ≥3×10 9CFU / mL, the suspension rate is more than 80%, and the pH is 6.0-7.0. The bacillus Bs916 seed treatment agent can promote the germination of rice seeds and the growth of seedlings, and has a good control effect on rice seedling disease. When the bacillus Bs916 seed treatment agent is mixed with tebuconazole (250 mg / L), jingangmycin (50 mg / L) and nongkang 120 (200 mg / L) respectively, the control effects on rice seedling disease are 87.54%, 86.74% and 88.05% respectively, which are almost equivalent to the control effect of the commercial chemical pesticide “bright shield (oxadixyl + fludioxonil)”. In summary, the application discloses a microbial seed treatment agent which has the functions of promoting growth and controlling rice seedling disease. The seed treatment agent can be mixed with chemical pesticides to greatly reduce the amount of chemical pesticides, reduce pesticide residues, and has great application value in agricultural production and market conversion development prospects. SUMMARY

[0006] The application aims to provide a microbial seed treatment agent for controlling rice seedling disease, which is characterized in that the microbial seed treatment agent contains active ingredient bacillus subtilus Bs916 with a preservation number of CGMCC No.0808. The microbial seed treatment agent is a red viscous suspension liquid, and the content of living bacteria (spores) is greater than or equal to 3×10 9 CFU / mL, the suspension rate is more than 80%, and the pH is 6.0-7.0.

[0007] The active strain bacillus subtilus Bs916 of the microbial seed treatment agent has a strong effect of inhibiting the pathogenic fusarium of rice seedling disease, and has a strong inhibitory effect on the mycelial growth of fusarium fujikuroi, fusarium proliferatum, fusarium graminearum and fusarium asiaticum.

[0008] The application provides a preparation formula of a microbial seed treatment agent, and the effective active ingredient of the microbial seed treatment agent is composed of spore fermentation liquor of bacillus subtilus Bs916 in YPG culture medium, and the main ratio of the preparation formula is as follows: the content of living bacteria (spores) of the strain Bs916 is greater than or equal to 3×10 9 CFU / mL, a film forming agent (polyvinyl alcohol 3-5%, w / v), a thickening agent (sodium alginate 0.5-1%, w / v), a dispersing agent (xanthan gum 0.1-0.2%, w / v), an antifreezing agent (1,2-propanediol 5-10%, w / v), a warning color (acid fuchsin 0.6-1%, w / v), and a preservative (sodium benzoate 0.05-0.1%, w / v), and the pH is 6.0-7.0.

[0009] The microbial seed treatment agent of the present invention can be used to prevent and treat rice bakanae disease caused by a complex bacterial community of Fusarium fujikura, and the seed treatment agent has the effect of promoting seed germination and seedling growth.

[0010] The present invention discloses a microbial seed treatment agent for preventing and treating rice seedling disease, as well as a preparation method and application examples. The active ingredient of the microbial seed treatment agent is Bacillus subtilis Bs916, which has a good inhibitory effect on multiple species of the Fusarium fujikura complex. The Bacillus subtilis Bs916 seed treatment agent can be used alone or in combination with commercially available pesticides (tebuconazole, jinggangmycin, and agricultural anti-120) to prevent and treat rice seedling disease caused by the Fusarium fujikura complex. The seed treatment agent also has a certain growth-promoting effect and is highly valuable for application in agricultural production and has great prospects for market transformation and development.

[0011] The present invention is further described below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Inhibitory effect of Bacillus sp. Bs916 on different species of Fusarium spp. causing rice bakanae disease on plates

[0013] Figure 2 Effects of Bacillus sp. Bs916 seed treatment agent mixed with tebuconazole on rice seed germination

[0014] Figure 3 Growth of rice seedlings after treatment with Bacillus sp. Bs916 seed treatment agent mixed with tebuconazole

[0015] Figure 4 Effects of Bacillus sp. Bs916 seed treatment agent mixed with Jinggangmycin on rice seed germination

[0016] Figure 5 Growth of rice seedlings after treatment with Bacillus subtilis Bs916 seed treatment agent mixed with Jinggangmycin DETAILED DESCRIPTION

[0017] Example 1 Inhibitory Effect of Bacillus sp. Bs916 on Different Rice Bakanae Disease Pathogens

[0018] Research has shown that rice bakanae disease is caused by multiple Fusarium species, primarily the Fusarium fujikura complex, which includes Fusarium fujikuroi, Fusarium proliiferatum, Fusarium asiaticum, and Fusarium graminearum. The inventors of this patent used a plate standoff assay to determine the inhibitory activity of the Bs916 strain against six different species of rice bakanae disease. The specific steps are as follows:

[0019] The test pathogen strains included F. fufujikuroi JR-3 and GY5b, F. proliferatum 2JR-3 and RGZ-1, F. asiaticum 25-2, and F. graminearum DG-9, all maintained by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences. Fungi were cultured on PDA medium (200 g potato, 20 g glucose, 20 g agar, and 1000 mL distilled water, pH 6.5–7.0) at 28°C for 5 days and then used.

[0020] Bacillus subtilus Bs916 was activated on LB medium, then transferred to 50 mL of LB broth and cultured at 28°C with shaking (150 rpm / min) for 2 days before use. A 5 mm diameter cake of the plant pathogenic fungus was placed in the center of a blank PDA plate. With the pathogen at the center, 5 μL of the Bs916 solution was dripped 2.5 cm from the pathogen. Three replicates were run for each treatment. An LB broth control was included. The plates were incubated at 26°C, and the antibacterial effect of the antagonist against the pathogen was assessed when the control plate was fully colonized.

[0021] The results of the investigation on the inhibition bandwidth are as follows Figure 1 As shown, Bs916 had the best inhibitory effect against F. asiaticum 25-2, with an average inhibition band of 14.75 mm. The average inhibition bands against F. fujikuroi JR-3 and GY5b were 12.25 mm and 10.5 mm, respectively. The average inhibition bands against F. proliferatum 2JR-3 and RGZ-1 were 10.5 mm and 8.0 mm, respectively. The strain had the worst inhibitory effect against F. graminearum DG-9, with an average inhibition band of 6.0 mm. These results indicate that strain Bs916 has good inhibitory effects against different species of Fusarium oxysporum, the pathogenic fungus of rice bakanae disease, but there are significant differences between different strains.

[0022] Example 2 Preparation of Bacillus subtilis Bs916 seed treatment agent

[0023] Preparation of Bacillus subtilis Bs916 spore fermentation broth: First, activate a single colony of Bacillus subtilis Bs916 on a YPG plate. Then, inoculate the single colony into YPG liquid medium and culture overnight at 28°C, 150 rpm, in a shaker to form a fermentation seed broth. This seed broth was inoculated at a 1% inoculum into a 330 ml / 1 L fermentation flask and shaken until at least 85% of the cells formed spores. The spore content was then measured. The YPG medium recipe is as follows: 5 g yeast powder, 5 g peptone, 5 g glucose, dilute to 1 L with distilled water, pH 7.0. Add 20 g agar if necessary.

[0024] Preparation of Bacillus subtilis Bs916 seed treatment agent: Bacillus subtilis Bs916 seed treatment agent was prepared according to the formula of microbial seed treatment agent. The specific process was as follows: Bs916 spore fermentation liquid was placed in a preparation container at a volume ratio of 50%, and 0.1% preservative, 3% film-forming agent, 1% thickener, 0.1% dispersant, 5% antifreeze agent, and 1% warning color were added at the same time. The mixture was stirred evenly, the pH was adjusted to 6.0-7.0, and water was added to make the volume 1L, thus forming Bacillus Bs916 seed treatment agent (spore content ≥3×10 9 CFU / mL). The seed treatment agent samples met the standards shown in Table 1. After seed dressing, the seeds were brightly and uniformly colored.

[0025] The microbial seed treatment agent formulation followed in this example is as follows: microbial fermentation broth (50-70%), film-forming agent (polyvinyl alcohol 3-5%, w / v), thickener (sodium alginate 0.5-1%, w / v), dispersant (xanthan gum 0.1-0.2%, w / v), antifreeze agent (1,2-propylene glycol 5-10%, w / v), warning color (acid fuchsin 0.6-1%, w / v) and preservative (sodium benzoate 0.05-0.1%, w / v), and finally water is added to make the volume 1 L.

[0026] Table 1 Main index parameters of Bacillus sp. Bs916 seed treatment agent sample product

[0027]

[0028] Example 3 Effect of Bs916 seed treatment agent mixed with tebuconazole on preventing and controlling rice bakanae disease

[0029] Tebuconazole is a common commercially available chemical pesticide, primarily used to control plant fungal diseases. It has a wide range of applications and is being used in increasing quantities. Based on the guiding principle of reducing chemical pesticide usage while simultaneously improving the stable and efficient field efficacy of live microbial pesticides, this study evaluated the efficacy of a Bacillus sp. Bs916 seed treatment agent mixed with varying concentrations of tebuconazole against rice bakanae disease. The Bacillus sp. Bs916 seed treatment agent used in this experiment is provided in Example 2.

[0030] This experiment was carried out in 2022 at the Lishui Plant Science Experimental Base of Jiangsu Academy of Agricultural Sciences. The rice variety was Nanjing 46, and the soil type was loam with uniform fertility. On May 1, the rice seeds were coated with a drug-seed ratio of 1:50, and stirred thoroughly until the drug solution was evenly distributed on the surface of the seeds. After drying, they were directly germinated and sown. The rice seedlings were transplanted on June 8, and the planting density of the nursery field was 12,000 holes / mu. The experimental plots were surrounded by rice, and the rice in each plot grew well and evenly. Fertilizer and water management were carried out as usual. No other fungicides for the prevention and control of rice seedling disease were applied during the experiment. There were 8 treatments in the experiment. The specific agent design and concentration are shown in Table 2. The rice seedlings were raised at 8-10m per agent treatment. 2 After sowing and transplanting into the field, each agent was treated 4 times, with each treatment lasting 150m 2 After seed dressing, a laboratory constant-temperature germination test (28°C) was conducted with 100 seeds per treatment replicated three times. Germination rates were determined three days later. Furthermore, seedling bud growth was observed regularly and photographed on the ninth day. During the heading stage in the field, five random surveys were conducted at each plot, with 1,000 plants surveyed at each site. The number of diseased plants was recorded, the incidence rate was calculated, and the control efficacy was calculated using the following formula.

[0031] Diseased plant rate (%) = (number of diseased plants / total number of plants surveyed) × 100

[0032] Control effect (%) = [(blank control area disease rate - treatment area disease rate) / blank control area disease rate] × 100

[0033] The test agents and their concentrations are shown in Table 2:

[0034] Table 2 Test design and concentration of test agents

[0035]

[0036] The experimental data of the effect of Bs916 seed treatment agent mixed with tebuconazole on rice seed germination showed that ( Figure 2 ): The rice seeds treated with a mixture of Bs916 and tebuconazole (250 mg / L) had the highest germination rate of 91.00%, followed by the seeds treated with Bs916 alone with a germination rate of 89.00%. The rice seeds treated with "Liangdun", a commonly used product for preventing and treating rice bakanae disease, had a germination rate of 88.00%, all of which were higher than the germination rate of the water control treatment (87.00%). The rice seeds treated with tebuconazole (1 g / L) and prochloraz alone had germination rates of 86.00% and 84.67%, respectively, which were significantly lower than the control treatment. Further observation of the effects of each treatment on the growth of rice seedlings showed that the rice seedlings had a germination rate of 89.00%. Figure 3The results showed that the commercially available chemical pesticides "Liangdun", myclobutanil and tebuconazole, when used alone, had varying degrees of inhibitory effects on the growth of rice seedlings. However, when the Bs916 seed treatment agent was used alone or mixed with tebuconazole, the growth of rice seedlings was slightly increased compared with the control.

[0037] Results of a field investigation into the efficacy of Bs916 seed treatment against rice bakanae disease at the heading stage (Table 3) show that Bs916 seed treatment alone achieved a 70.25% efficacy against bakanae disease. Tebuconazole (1 g / L) and prochloraz alone achieved efficacy of 83.37% and 80.32%, respectively. When Bs916 seed treatment was mixed with varying concentrations of tebuconazole, its efficacy against bakanae disease increased to 84.00%-88.00%. The combination of Bs916 seed treatment and tebuconazole (250 mg / L) achieved an efficacy of 87.54%, which was not significantly different from the efficacy of Liangdun, a commonly used commercially available product for controlling rice bakanae disease (87.64%). The combined results of these studies indicate that the combination of Bs916 and tebuconazole promotes both rice seed germination and seedling growth, mitigating the inhibitory effects of tebuconazole alone. Furthermore, the combined treatment of Bs916 and tebuconazole (250 mg / L) demonstrated comparable efficacy to the commonly used commercial product "Liangdun," but with a 75% reduction in tebuconazole dosage.

[0038] Table 3 Effect of Bs916 seed treatment agent mixed with tebuconazole on the control of rice bakanae disease (heading stage)

[0039]

[0040] In the significant difference analysis, lowercase letters represent P < 0.05 level, and uppercase letters represent P < 0.01 level.

[0041] Example 4 Effect of Mixing Bs916 Seed Treatment Agent and Jinggangmycin in Preventing and Controlling Rice Bakanae Disease

[0042] Jinggangmycin is a common commercially available antibiotic biopesticide, primarily used to control fungal plant diseases such as rice sheath blight. Based on the principle of enhancing the field efficacy and stability of live microbial pesticides, this study evaluated the efficacy of Bacillus sp. Bs916 seed treatment mixed with varying concentrations of Jinggangmycin against rice bakanae disease. The Bacillus sp. Bs916 seed treatment used in this experiment is provided in Example 2.

[0043] This experiment was carried out in 2023 at the Lishui Plant Science Experimental Base of Jiangsu Academy of Agricultural Sciences. The rice variety was Nanjing 46, and the soil type was loam with uniform fertility. On May 9, the rice seeds were coated with a drug-seed ratio of 1:50, and stirred thoroughly until the drug solution was evenly distributed on the surface of the seeds. After drying and germination, they were directly sown. The experimental plots were surrounded by rice. The rice in each plot grew well and uniformly. Fertilizer and water management were carried out as usual. No other fungicides for the prevention and control of rice seedling disease were applied during the experiment. A total of 8 treatments were set up in the experiment. The specific drug design and concentration are shown in Table 4. Each drug treatment was repeated 4 times, and each repetition was 150m 2 After seed dressing, a laboratory constant-temperature germination test (28°C) was conducted with 100 seeds per treatment replicated three times. Germination rates were determined three days later. Furthermore, seedling bud growth was observed regularly and photographed on the ninth day. During the heading stage in the field, five random surveys were conducted at each plot, with 1,000 plants per site. The number of diseased plants was recorded, the incidence rate was calculated, and the control efficacy was calculated using the following formula.

[0044] Diseased plant rate (%) = (number of diseased plants / total number of plants surveyed) × 100

[0045] Control effect (%) = [(blank control area disease rate - treatment area disease rate) / blank control area disease rate] × 100

[0046] The test agents and their concentrations are shown in Table 4:

[0047] Table 4 Test design and concentration of test agents

[0048]

[0049] The experimental data of the effect of Bs916 seed treatment agent mixed with Jinggangmycin on rice seed germination showed that ( Figure 4 ): The seed germination rate of the control treatment was 89.67%, the seed germination rate of the Bs916 seed treatment agent alone was 90.33%; the seed germination rate of the single-use jinggangmycin (50 mg / L) was 91.00%. The germination rates of rice seeds treated with a mixture of Bs916 and jinggangmycin (10 mg / L, 25 mg / L, 50 mg / L) were 86.33%, 90.00% and 88.33%, respectively. The germination rates of rice seeds treated with the commercially available products Liangdun and prochloraz were 88.00% and 87.00%, respectively, which were significantly lower than those of the other treatments. Further observation of the effects of each treatment on the growth of rice seedlings showed the following results: Figure 5 As shown in the figure: The commercially available chemical pesticides "Liangdun" and myclobutanil have varying degrees of inhibitory effects on the growth of rice seedlings, while the Bs916 seed treatment agent has no significant inhibitory effect on the growth of rice seedlings when used alone or mixed with jinggangmycin, and has a slight promoting trend.

[0050] Results of a field investigation into the efficacy of Bs916 seed treatment against bakanae disease at the heading stage (Table 5) show that Bs916 seed treatment alone achieved a 76.69% efficacy against bakanae disease. When Bs916 was mixed with varying concentrations of jinggangmycin, its efficacy increased to 79.00%-87.00%. The efficacy of Bs916 mixed with jinggangmycin (50 mg / L) was 86.74%, lower than the efficacy of the commercially available product "Liangdun" (90.95%), but significantly higher than the efficacy of prochloraz (79.73%). These results suggest that the combination of Bs916 seed treatment and jinggangmycin had no significant inhibitory effect on rice seed germination and seedling growth, but significantly improved its efficacy against bakanae disease compared to either agent alone.

[0051] Table 5 Effect of Bs916 seed treatment agent mixed with tebuconazole on the prevention and control of rice bakanae disease (heading stage)

[0052]

[0053] In the significant difference analysis, lowercase letters represent P < 0.05 level, and uppercase letters represent P < 0.01 level.

[0054] Example 5 Effect of Mixing Bs916 Seed Treatment Agent and Agricultural Antibiotic 120 in Preventing and Controlling Rice Bakanae Disease

[0055] Agroantibiotic 120, a nucleoside pyrimidine antibiotic, is a commercially available biopesticide primarily used to control fungal plant diseases. Based on the principle of enhancing the field efficacy and stability of live microbial pesticides, this study evaluated the efficacy of Bacillus sp. Bs916 seed treatment mixed with varying concentrations of Agroantibiotic 120 against rice bakanae disease. The Bacillus sp. Bs916 seed treatment used in this experiment is provided in Example 2.

[0056] This experiment was carried out in 2023 at the Yangzhou Rice Experimental Base in Jiangsu Province. The rice variety was Ningxiangjing. The soil and fertilizer were evenly distributed. The rice seeds were coated with the medicine-seed ratio of 1:50 on May 20, and stirred thoroughly until the medicine liquid was evenly distributed on the surface of the seeds. They were dried and sown. The rice seedlings were transplanted on June 15. The rice in each plot of the experiment grew well and evenly. Fertilizer and water management were carried out as usual. No other fungicides for the prevention and control of rice seedling disease were applied during the experiment. There were 3 treatments in the field control efficacy test. The specific drug design and concentration are shown in Table 6. Each drug treatment was repeated 4 times, and each repetition was 150m 2 During the heading period, 5 random survey points were selected in each plot, with 1,000 plants at each point. The number of diseased plants was recorded, the incidence rate was calculated, and the control effect was calculated according to the following formula.

[0057] Diseased plant rate (%) = (number of diseased plants / total number of plants surveyed) × 100

[0058] Control effect (%) = [(blank control area disease rate - treatment area disease rate) / blank control area disease rate] × 100

[0059] The seed germination rate test was carried out in the laboratory. There were 4 treatments in total (Table 6). After the seeds were mixed with the pesticide, 3 replicates were performed for each treatment, and 100 seeds were used in each replicate to conduct a laboratory constant temperature germination test (28°C). After 3 days, the seed germination rate was statistically determined.

[0060] Table 6 Test design and concentration of test agents

[0061]

[0062]

[0063] Experimental data on the effect of a combination of Bs916 seed treatment and Agricultural Antibiotic 120 on rice seed germination (Table 7) show that the germination rate of the untreated control was 61.33%, while that of seeds treated with Bs916 alone was 77.67%. The germination rates of rice seeds treated with a combination of Bs916 seed treatment and 100 and 200 mg / L Agricultural Antibiotic 120 were 69.67% and 70.67%, respectively, with no significant difference between the two treatments. These rates were significantly higher than the untreated control, but significantly lower than those treated with Bs916 seed treatment. Field studies on the efficacy of Bs916 seed treatment against bakanae disease at the heading stage are shown in Table 7: The combined efficacy of Bs916 seed treatment and 100 and 200 mg / L Agricultural Antibiotic 120 against bakanae disease was 67.47% and 88.05%, respectively. These results demonstrate that Bs916 seed treatment can promote rice seed germination and alleviate the inhibitory effects of Agro-Antibiotic 120 on rice seed germination. Mixing Bs916 with 200 me / L Agro-Antibiotic 120 significantly enhances its efficacy against rice bakanae disease by 88.05%.

[0064] Table 7 Effects of mixed application of Bs916 seed treatment agent and agricultural anti-120 on rice seed germination and control of rice bakanae disease (heading stage)

[0065]

[0066] In the significant difference analysis, lowercase letters represent P < 0.05 level, and uppercase letters represent P < 0.01 level.

Claims

1. The present invention discloses a microbial seed treatment agent for preventing and treating rice bakanae disease, characterized in that: The microbial seed treatment agent contains the active ingredient Bacillus subtilus Bs916, with a deposit number of CGMCC No. 0808. This strain has a strong inhibitory effect on various rice seedling disease-causing Fusarium spp. The microbial seed treatment agent appears as a red viscous suspension with a live bacteria (spore) content of ≥3×10 9 CFU / mL, the suspension rate is more than 80%, and the pH is 6.0-7.

0.

2. The microbial seed treatment agent according to claim 1, wherein The seed treatment agent includes various auxiliary agents, namely a film-forming agent (polyvinyl alcohol 3-5%, w / v), a thickener (sodium alginate 0.5-1%, w / v), a dispersant (xanthan gum 0.1-0.2%, w / v), an antifreeze agent (1,2-propylene glycol 5-10%, w / v), a warning color (acid fuchsin 0.6-1%, w / v), and a preservative (sodium benzoate 0.05-0.1%, w / v).

3. The method for preparing the microbial seed treatment agent according to claim 1 or 2, wherein: 3.1 Liquid fermentation medium is YPG medium with the following formula: 5 g yeast powder, 5 g peptone, 5 g glucose, distilled water to 1 L, pH 7.0, and 20 g agar if solids are needed; 3.2 Preparation of Bacillus Bs916 spores, the active ingredient of the microbial seed treatment agent: First, activate a single colony of Bacillus Bs916 on a YPGA plate. Subsequently, the single colony was inoculated into YPG liquid culture medium and cultured overnight in a shaking incubator at 28°C and 150 rpm to form a fermentation seed liquid. The seed liquid was inoculated into a 330 ml / 1 L fermentation flask at a 1% inoculation rate and cultured at 28°C and 150 rpm with shaking until more than 85% of the bacteria formed spores, and the fermentation spore content was detected. 3.3 Preparation of microbial seed treatment agent: The fermented spore solution was placed into the agent preparation tank at a volume ratio of 50%, and 0.1% preservative, 3% film-forming agent, 1% thickener, 0.1% dispersant, 5% antifreeze agent, and 1% warning color were added at the same time. The mixture was stirred evenly and the pH was adjusted to 6.0-7.0 to form a microbial seed treatment agent containing Bacillus sp. Bs916 (spore content ≥ 3×10 9 CFU / mL).

4. Use of the microbial seed treatment agent prepared according to claim 3 in rice planting, characterized in that: The seed treatment agent is mixed with dry rice seeds or newly whitened seeds at a ratio of 1:30-1:50 (v / w) until the red color is evenly distributed on the surface of the seeds. The seeds can be sown after being dried in the shade.

5. The microbial seed treatment agent prepared according to claim 3, when used alone for seed dressing, has the effects of promoting rice seed germination and seedling growth and preventing and controlling rice bakanae disease.

6. The microbial seed treatment agent prepared according to claim 3 is mixed with commercially available pesticides to achieve a higher efficacy and better stability in preventing and controlling rice bakanae disease, and can significantly reduce the amount of chemical pesticides used, thereby reducing the harm caused by pesticide residues. When the Bacillus sp. Bs916 seed treatment agent is mixed with tebuconazole (250 mg / L), jinggangmycin (50 mg / L), and agricultural antibiotic 120 (200 mg / L), the efficacy of preventing and controlling rice bakanae disease is 87.54%, 86.74%, and 88.05%, respectively, and the efficacy is stable.

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