A composite bactericide containing bacillus velezensis and application thereof

Abstract

The application relates to a composite fungicide containing bacillus velezensis and an application thereof and belongs to the technical field of biotechnology. In order to solve the technical problems of limited bacteriostasis, high environmental sensitivity and slow effect of springomycin or bacillus velezensis in biological prevention, the application provides bacillus velezensis KN-901, and a composite fungicide containing the bacillus velezensis KN-901 and springomycin, wherein the preservation number of the bacillus velezensis KN-901 is CCTCC M 20242442. The composite fungicide provided by the application has the double action mechanisms of springomycin and bacillus velezensis, has the synergistic effect, has a good control effect on plant fungal or bacterial diseases, has a certain yield-increasing effect on plants, and can be widely applied to the prevention of field plant diseases and yield increase.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a compound fungicide containing bacillus velezensis and application thereof. BACKGROUND

[0002] Plant diseases have been one of the major problems affecting agricultural production. The traditional prevention and control method is to use chemical pesticides for prevention and control. Although the diseases can be effectively prevented and controlled, long-term overuse can easily lead to environmental pollution and enhanced pathogen resistance. As a new type of environmentally friendly biological pesticide, microbial agents have the advantages of no residue, no pollution, and not prone to drug resistance.

[0003] Springomycin is an aminoglycoside antibiotic that can interfere with the amino acid synthesis system of prokaryotic or eukaryotic organisms, has the advantages of low toxicity, low residue, and strong systemicity, and can produce good fungicidal effect. Bacillus velezensis is a new species of Bacillus, which has more excellent ability to produce secondary metabolites and colonize plants. Therefore, it has attracted more attention. As a beneficial microorganism with broad-spectrum fungicidal activity and the ability to promote plant growth, bacillus velezensis can not only inhibit plant diseases by producing extracellular hydrolases and antagonistic active substances, but also induce plant resistance and regulate plant rhizosphere microbial flora to inhibit the growth of pathogenic bacteria.

[0004] In the prior art, springomycin and bacillus velezensis are widely used in agricultural production as independent pesticides and biological control agents. However, there are certain limitations when they are used alone, such as limited inhibition of springomycin on specific pathogenic bacteria, high environmental sensitivity of bacillus velezensis, and slow effect. In addition, there is a lack of microbial agent products that complexly use the two in the market, which cannot meet the diversified needs in agricultural production. Therefore, those skilled in the art are eager to develop a biological control agent that can inhibit a variety of plant pathogenic bacteria, produce a variety of metabolites, prevent and control plant diseases quickly and effectively, induce plants to produce self-resistance, and achieve the effect of promoting growth and yield. SUMMARY

[0005] The present application provides a compound fungicide containing bacillus velezensis and application thereof to solve the technical problems of limited fungicidal effect, high environmental sensitivity, and slow effect when springomycin or bacillus velezensis is used alone for biological control in the prior art, and the technical blank of lacking complex biological control of the two.

[0006] One of the purposes of the present application is to provide a compound fungicide, wherein the effective components of the compound fungicide are fermentation broth and / or bacterial powder of springomycin and bacillus velezensis.

[0007] In a preferred embodiment of the present application, the mass percentage of kasugamycin in the fungicide is 1-10%; the viable bacterial count of Bacillus velezensis fermentation liquor and / or bacterial powder in the fungicide is 10-100 billion CFU / g; and the dosage form of the fungicide is any one of the suspension concentrate or the wettable powder.

[0008] In a preferred embodiment of the present application, when the dosage form of the fungicide is the suspension concentrate, the suspension concentrate further comprises xanthan gum, silicone and sodium alginate; the viable bacterial count of Bacillus velezensis fermentation liquor and / or bacterial powder in the suspension concentrate is 10-100 billion CFU / g; the mass percentages of kasugamycin, xanthan gum, silicone and sodium alginate in the suspension concentrate are 2%-5%, 1%-5%, 2%-5% and 2%-6% respectively; and the application amount of the suspension concentrate is 60-120 mL / acre.

[0009] In a preferred embodiment of the present application, when the dosage form of the fungicide is the wettable powder, the wettable powder further comprises sodium lignosulfonate, sodium polynaphthaleneformaldehyde sulfonate and kaolin; the viable bacterial count of Bacillus velezensis fermentation liquor and / or bacterial powder in the wettable powder is 200-1000 billion CFU / g; the mass percentages of kasugamycin, sodium lignosulfonate, sodium polynaphthaleneformaldehyde sulfonate and kaolin in the wettable powder are 5%-10%, 4%-6%, 1%-3% and 30%-70% respectively; and the application amount of the wettable powder is 40-80 g / acre.

[0010] In a preferred embodiment of the present application, the Bacillus velezensis is Bacillus velezensis KN-901, the preservation number of which is CCTCC M 20242442; and the 16S rRNA sequence of the Bacillus velezensis KN-901 is shown in SEQ ID NO. 1.

[0011] The second object of the present application is to provide the application of the above-mentioned composite fungicide in preventing and treating plant fungal diseases and bacterial diseases.

[0012] The third object of the present application is to provide the application of the above-mentioned composite fungicide in increasing the yield of plants.

[0013] The fourth object of the present application is to provide a Bacillus velezensis KN-901, the preservation number of which is CCTCC M 20242442, the classification and name of which is Bacillus velezensis, which is preserved in the China Center for Type Culture Collection, and the preservation date of which is November 06, 2024.

[0014] In a preferred embodiment of the present application, the 16S rRNA sequence of Bacillus velezensis KN-901 is shown in SEQ ID NO. 1.

[0015] The fifth object of the present application is to provide the use of the above-mentioned Bacillus velezensis KN-901 and its fermentation product in the preparation of a plant fungicide.

[0016] The present application provides a composite fungicide containing Bacillus velezensis and its fermentation product and kasugamycin, and a Bacillus velezensis KN-901 with a preservation number of CCTCC M 20242442 and a 16S rRNA sequence shown in SEQ ID NO. 1. The composite fungicide provided by the present application takes the fermentation broth and / or bacterial powder of the above-mentioned Bacillus velezensis KN-901 and kasugamycin as the main effective components, and has good bacteriostatic effect on bacterial and fungal pathogenic bacteria. Compared with Bacillus velezensis single agent and common kasugamycin compound products in the market, the bacteriostatic effect of the composite fungicide is increased by 14-37% compared with Bacillus velezensis KN-901 single agent. The composite fungicide has more excellent bacteriostatic effect on difficult-to-control soil-borne diseases.

[0017] Field experiments show that the composite fungicide provided by the present application reduces the incidence of potato scab to 31.74%-42.56% and the disease index to 9.31-15.16 compared with the conventional treatment control group. When the use amount of the composite fungicide is 120 ml / acre, the control effect is as high as 78.71%. The composite fungicide has a significant yield-increasing effect on field crops, with a yield-increasing rate of 22.45%.

[0018] The composite fungicide provided by the present application reduces the disease index of yam anthracnose to 7.45-11.23 compared with the conventional treatment control group. When the use amount of the composite fungicide is 80 g / acre, the control effect is as high as 80.77%. The composite fungicide has a significant yield-increasing effect on field crops, with a yield-increasing rate of 29.58%.

[0019] The composite fungicide provided by the present application reduces the disease index of sugar beet root rot to 5.43-7.63 compared with the conventional treatment control group. When the use amount of the composite fungicide is 120 ml / acre, the control effect is as high as 74.84%. The composite fungicide has a significant yield-increasing effect on field crops, with a yield-increasing rate of 40.79% and a sugar content increase rate of 27.11%.

[0020] Compared with the conventional treatment control group, the complex fungicide provided by the present application reduces the disease index of rice blast to 2.49-4.06, and when the use amount of the complex fungicide is 80ml / mu, the control effect is as high as 89.48%, the complex fungicide has a significant synergistic effect on the yield of field crops, and the yield increase rate is 45.51%.

[0021] Compared with the conventional treatment control group, the complex fungicide provided by the present application reduces the disease index of bacterial fruit spot of Hami melon to 5.81-7.69, and when the use amount of the complex fungicide is 120ml / mu, the control effect is as high as 81.59%, the complex fungicide has a significant synergistic effect on the yield of field crops, and the yield increase rate is 36.38%.

[0022] The present application is prepared by compounding springomycin and bacillus velezensis, so as to play a double action mechanism, the springomycin has a wide bactericidal spectrum and fast effect, and the bacillus velezensis can produce antibacterial substances to inhibit pathogenic bacteria, has strong stress resistance and can induce plants to produce stress resistance, so as to synergistically improve the disease control effect.

[0023] The complex fungicide containing springomycin and bacillus velezensis provided by the present application has a significant effect in plant disease control, especially in the control effect on soil-borne diseases, which is significantly higher than that of springomycin single agent and bacillus velezensis single agent and the control effect of common springomycin compound fungicide on the market; the complex fungicide provided by the present application reduces the use of chemical pesticides, is more friendly to the environment, can delay the generation of pathogenic bacteria to single pesticide resistance, and can be widely applied to the control of field plant diseases.

[0024] The complex fungicide provided by the present application can be used with inorganic fungicides and organic fungicides in actual use process, and good control effects can be obtained in plant disease control; the inorganic fungicides are mainly divided into sulfur, copper and mercury fungicides; the organic fungicides are mainly divided into organic sulfur (such as mancozeb), trichloromethyl sulfenyl (such as captan), substituted benzene (such as chlorothalonil), pyrrole (such as fenpiclonil), organophosphorus (such as phosphorus aluminum), benzimidazole (such as carbendazim), triazole (such as triadimefon and triadimenol), phenylamide (such as metalaxyl) and the like. DETAILED DESCRIPTION

[0025] Those skilled in the art can improve the process parameters according to the content herein. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the related personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present application, to realize and apply the present application technology.

[0026] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with specific embodiments. The test methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0027] The springomycin used in the following examples is obtained by fermentation and isolation and purification by the company itself or purchased on the market.

[0028] The Bacillus velezensis KN-901 used in the following examples is obtained by isolation and purification from soil samples collected from a place where soil-borne diseases frequently occur in Xiangyang, Hubei, and is preserved. The preservation number is CCTCC M 20242442, the classification and naming is Bacillus velezensis, and the preservation date is November 06, 2024. The preservation center is the China Center for Type Culture Collection, and the preservation date is November 06, 2024.

[0029] Example 1: Detection of the antibacterial activity of springomycin and different Bacillus velezensis on pathogenic bacteria

[0030] The pathogenic bacteria used in this example include fungi and bacteria. The fungi include: yam anthracnose-Colletotrichum gloeosporioides, beet root rot 1-Rhizoctonia solani, rice blast-Magnaporthe grisea; the bacteria include: potato scab-Streptomyces scabiei, beet root rot 2-Erwinia carotovora subsp. betivora, cucumber bacterial angular spot-Pseudomonas syringae. The above strains are obtained by isolation and purification by the company from the corresponding diseased soil.

[0031] The Bacillus velezensis BNCC-364390, CCTCC AB 2016194, ACCC 03040 used in this example are purchased from Beina Biological Technology Co., Ltd., China Center for Type Culture Collection, and China Agricultural Microbial Culture Collection and Preservation Center, respectively.

[0032] S1: Activation of pathogenic bacteria: Take the yam anthracnose-colicolletotrichum, beet root rot 1- rhizoctonia solani, rice blast-gray pyricularia, potato scab-streptomyces scabiei, beet root rot 2- erwinia carotovara subsp. betivora and cucumber bacterial angular spot-pseudomonas syringae species preserved in the ultra-low temperature refrigerator, inoculate the above pathogenic bacteria on PDA / NA plates, and culture at 28℃ for 5-7d (different pathogenic bacteria have different culture times), pick the mycelium on the culture plate of the cultured fungal pathogenic bacteria and inoculate into PDA liquid medium (add crushed glass slag), and shake culture at 28℃, 160rpm for 2d to obtain fungal bacterial suspension respectively; pick the mycelium on the culture plate of the cultured bacterial pathogenic bacteria and inoculate into ISP2 / LB liquid medium, and shake culture at 30℃, 160rpm for 24h to obtain bacterial bacterial suspension of yam anthracnose-colicolletotrichum, beet root rot 1- rhizoctonia solani, rice blast-gray pyricularia, potato scab-streptomyces scabiei, beet root rot 2- erwinia carotovara subsp. betivora and cucumber bacterial angular spot-pseudomonas syringae respectively;

[0033] S2: Preparation of spring ray mycin: 70% spring ray mycin raw drug is configured into 6% spring ray mycin solution for standby;

[0034] S3: Preparation of bacillus velezensis fermentation broth: The bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194 and ACCC 03040 preserved in the ultra-low temperature refrigerator are inoculated into LB liquid medium respectively, and shake culture at 30℃, 160rpm for 24h, the seed liquid of the cultured bacillus velezensis is inoculated into the sterilized fermentation medium respectively, and culture at 30℃, 260rpm for 40-50h to obtain the fermentation broth of bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194 and ACCC 03040 respectively, and the viable spore number of the fermentation broth is about 10-15 billion CFU / ml;

[0035] The fermentation medium: soybean meal 25g / L, starch 30g / L, yeast extract 8g / L, corn syrup 15g / L, peptone 5g / L, calcium chloride 0.25g / L, potassium dihydrogen phosphate 0.18g / L, magnesium sulfate heptahydrate 0.36g / L, manganese chloride 0.45g / L;

[0036] S4: Inhibition activity was determined by Oxford cup method: bacterial bacterial suspension of S1 obtained of Colletotrichum gloeosporioides, Rhizoctonia solani, Pyricularia grisea, Streptomyces scabies, Erwinia carotovora subsp. betivorum and Pseudomonas syringae was added to PDA / NA agar medium, mixed evenly, then poured flat, Oxford cups (8mm in diameter) were placed on four areas of the flat (90mm), 150μL of 6% kasugamycin solution obtained in S2, and fermentation broth of Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194 and ACCC 03040 obtained in S3 were added into the Oxford cups, and water was added as a control, cultured for 24-48h, whether to produce inhibition zone was observed, and the diameter of the inhibition zone was measured by vernier caliper.

[0037] Inhibition: no inhibition for inhibition zone <10mm, moderate inhibition for 10mm < inhibition zone <15mm, and high inhibition for inhibition zone >15mm; the outer diameter of the Oxford cup was 8mm, the larger the inhibition zone, the better the inhibition effect, and the inhibition zone referred to the diameter of the entire circular transparent zone.

[0038] The results are shown in Table 1, and kasugamycin and Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194 and ACCC 03040 alone had certain inhibition effect on the growth of Streptomyces scabies, Colletotrichum gloeosporioides, Rhizoctonia solani, Erwinia carotovora subsp. betivorum, Pyricularia grisea and Pseudomonas syringae; and the Bacillus velezensis KN-901 had more excellent inhibition effect on the above pathogens than the Bacillus velezensis BNCC-364390, CCTCC AB 2016194 and ACCC 03040.

[0039] Table 1

[0040]

[0041] Example 2: Inhibition activity detection of kasugamycin and different Bacillus velezensis

[0042] S1: Bacillus velezensis sample preparation: Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194, ACCC 03040 stored in an ultra-low temperature refrigerator were inoculated on LB liquid medium, and cultured at 30°C, 160 rpm for 24 h. The seed liquid of the cultured Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194, ACCC 03040 was inoculated in the sterilized fermentation medium, and cultured at 30°C, 260 rpm for 40-50 h to obtain the fermentation liquid of Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194, ACCC 03040, and the viable spore number of the fermentation liquid was about 10-15 billion CFU / ml;

[0043] S2: Preparation of kasugamycin: 70% kasugamycin technical was configured into 3% and 6% kasugamycin solutions for standby;

[0044] S3: Preparation of composite fungicides: the fermentation liquid of Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194, ACCC 03040 obtained in S1 was respectively compounded with 6% kasugamycin obtained in S2 to obtain four kinds of composite fungicides;

[0045] S4: Oxford cup method was used to determine the inhibition activity: bacterial bacterial suspension of yam anthracnose-colicototrichum gloeosporioides, beet root rot 1- rhizoctonia solani, rice blast- pyricularia grisea, potato scab- streptomyces scabiei, beet root rot 2- erwinia carotovora subsp. betivora, and cucumber bacterial angular spot-pseudomonas syringae was respectively added to PDA / NA agar medium, and then mixed uniformly and poured flat. Oxford cups (8 mm in diameter) were placed on four areas of the flat plate (90 mm), 150 μL of 3% and 6% kasugamycin obtained in S2, four kinds of composite fungicides obtained in S1, and kasugamycin-mesnig and kasugamycin-bromoxynil samples (the 3% kasugamycin-2% mesnig and 2% kasugamycin-25% bromoxynil were purchased from the market) were added into the Oxford cups, and water was added as a control. After 24-48 h of culture, whether the inhibition zone was produced was observed, and the diameter of the inhibition zone was measured by using a vernier caliper.

[0046] The results are shown in Table 2. Compared with kasugamycin and fermentation liquor of Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194 and ACCC 03040, the bacteriostatic effect of the compound bactericide obtained by compounding kasugamycin and the four strains of Bacillus velezensis on S. scabies, C. gloeosporioides, R. solani, E. carotovora subsp. betavasculium, M. grisea and P. syringae was improved to different degrees.

[0047] It can be seen that the combination of Bacillus velezensis and kasugamycin has a synergistic effect. The comprehensive inhibition effect of the compound bactericide obtained by compounding Bacillus velezensis KN-901 and kasugamycin on the above pathogenic bacteria is significantly higher than that of the compound bactericide obtained by compounding Bacillus velezensis BNCC-364390, CCTCC AB 2016194 and ACCC 03040 and kasugamycin.

[0048] The compound bactericide containing kasugamycin and Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194 or ACCC 03040 provided by the present application significantly improves the bacteriostatic effect on S. scabies, C. gloeosporioides, R. solani, E. carotovora subsp. betavasculium, M. grisea and P. syringae compared with the existing bacteriostatic product Kasugamycin and Bromocyclen.

[0049] Table 2

[0050]

[0051] Example 3: Bacillus spore stability test of kasugamycin compounded with different Bacillus spores

[0052] S1: Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194, ACCC 03040 and Bacillus subtilis (obtained by the company itself) preserved in an ultra-low temperature refrigerator were inoculated on LB liquid medium respectively, and cultured at 30°C, 160 rpm for 24 h. The seed liquid of the cultured Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194, ACCC 03040 and Bacillus subtilis was inoculated into sterilized fermentation medium respectively, and cultured at 30°C, 260 rpm for 40-50 h to obtain Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194, ACCC 03040 fermentation liquid and Bacillus subtilis fermentation liquid, and the viable spore count of the fermentation liquid was about 10-15 billion CFU / ml;

[0053] S2: Preparation of spring thunder: 70% spring thunder raw drug was configured into 3% spring thunder solution for standby;

[0054] S3: Preparation of composite fungicide: the Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194, ACCC 03040 fermentation liquid and Bacillus subtilis fermentation liquid obtained in S1 were respectively compounded with 3% spring thunder obtained in S2 to obtain five kinds of composite fungicides.

[0055] In this embodiment, the composite fungicides containing Bacillus velezensis KN-901, BNCC-364390, CCTCC AB 2016194, ACCC 03040 and Bacillus subtilis respectively obtained above were sealed and stored in an environment of 22°C. The viable spore count was detected at 0, 3, 6, 12 and 18 months after storage. The detection method of viable spore count was plate counting method, and dilution and plate coating were performed after 20 min water bath at 80°C.

[0056] The results are shown in Table 3. After 18 months of compounding of Bacillus velezensis and spring thunder, the decrease rate of viable spore count of Bacillus velezensis in the composite fungicide was less than 10%. After 18 months of compounding of Bacillus subtilis and spring thunder, the decrease rate of viable spore count of Bacillus subtilis in the composite fungicide was as high as 52%. It can be seen that compared with Bacillus subtilis, Bacillus velezensis can maintain higher stability after compounding with spring thunder.

[0057] Table 3

[0058]

[0059] Example 4: Detection of the antibacterial activity of different complexing ratios of kasugamycin and Bacillus velezensis KN-901

[0060] S1: Bacillus velezensis KN-901 stored in an ultra-low temperature refrigerator was inoculated on LB liquid medium and cultured at 30°C, 160 rpm for 24 h. The cultured Bacillus velezensis KN-901 seed liquid was inoculated in sterilized fermentation medium and cultured at 30°C, 260 rpm for 40-50 h to obtain Bacillus velezensis KN-901 fermentation liquid, and the viable spore count of the fermentation liquid was about 10 billion CFU / ml;

[0061] S2: Preparation of kasugamycin: 70% kasugamycin technical material was configured into 3%, 6% and 9% kasugamycin solutions for standby;

[0062] S3: 3% kasugamycin obtained in S2 was complexed with Bacillus velezensis KN-901 fermentation liquid obtained in S1 to prepare complex bactericide 1, 6% kasugamycin obtained in S2 was complexed with Bacillus velezensis KN-901 fermentation liquid obtained in S1 to prepare complex bactericide 2, and 9% kasugamycin obtained in S2 was complexed with Bacillus velezensis KN-901 fermentation liquid obtained in S1 to prepare complex bactericide 3.

[0063] In this example, Oxford cup method was used to detect the antibacterial activity of Bacillus velezensis KN-901 fermentation liquid and complex bactericides 1-3 against Streptomyces scabies- Streptomyces scabies, Rhizoctonia solani- Rhizoctonia solani, Pseudomonas syringae- Pseudomonas syringae, and Cucumber bacterial angular spot- Pseudomonas syringae.

[0064] The results are shown in Table 4. The complex bactericides with different complexing ratios had certain synergistic effects on the colony inhibition of Streptomyces scabies- Streptomyces scabies, Rhizoctonia solani- Rhizoctonia solani, Pseudomonas syringae- Pseudomonas syringae, and Cucumber bacterial angular spot- Pseudomonas syringae relative to Bacillus velezensis KN-901 fermentation liquid, and the higher the concentration of kasugamycin in the complex bactericide, the better the inhibition effect on the pathogenic bacteria.

[0065] Table 4

[0066]

[0067] Example 5: Preparation of complex bactericide suspension

[0068] S1: Bacillus velezensis KN-901 preserved in a ultra-low temperature refrigerator was inoculated on LB liquid medium and cultured at 30°C, 160 rpm for 24 hours. The seed liquid of the cultured Bacillus velezensis KN-901 was inoculated in a sterilized fermentation medium and cultured at 30°C, 260 rpm for 60 hours to obtain a Bacillus velezensis KN-901 fermentation liquid, wherein the viable spore number of the fermentation liquid was about 15 billion CFU / ml;

[0069] The fermentation medium: soybean meal 25 g / L, starch 30 g / L, yeast extract 8 g / L, corn steep liquor 15 g / L, peptone 5 g / L, calcium chloride 0.25 g / L, potassium dihydrogen phosphate 0.18 g / L, magnesium sulfate heptahydrate 0.36 g / L, manganese chloride 0.45 g / L;

[0070] S2: 70% kasugamycin technical material was configured into a 3% kasugamycin solution for standby;

[0071] S3: the viable cell number of the Bacillus velezensis KN-901 fermentation liquid obtained in S1 was adjusted to 10 billion CFU / g. 3.1% kasugamycin obtained in S2, 3.5% silicone and 5% sodium alginate were sequentially added into the above adjusted Bacillus velezensis fermentation liquid, and then stirred and mixed uniformly. 3% xanthan gum was added, and stirred and mixed again uniformly. An acidity regulator was used to adjust the pH value to 4.5 to obtain a composite fungicide suspension agent.

[0072] Example 6: Preparation of a composite fungicide wettable powder

[0073] S1: Bacillus velezensis KN-901 preserved in a ultra-low temperature refrigerator was inoculated on LB liquid medium and cultured at 30°C, 160 rpm for 24 hours. The seed liquid of the cultured Bacillus velezensis KN-901 was inoculated in a sterilized fermentation medium and cultured at 30°C, 260 rpm for 60 hours to obtain a Bacillus velezensis KN-901 fermentation liquid, wherein the viable spore number of the fermentation liquid was about 15 billion CFU / ml;

[0074] The fermentation medium: soybean meal 25 g / L, starch 30 g / L, yeast extract 8 g / L, corn steep liquor 15 g / L, peptone 5 g / L, calcium chloride 0.25 g / L, potassium dihydrogen phosphate 0.18 g / L, magnesium sulfate heptahydrate 0.36 g / L, manganese chloride 0.45 g / L;

[0075] S2: the Bacillus velezensis KN-901 fermentation liquid obtained in S1 was filtered, centrifuged, dried and sprayed, and sieved to obtain Bacillus velezensis KN-901 spore powder;

[0076] S3: 70% kasugamycin technical material was configured into a 6% kasugamycin solution for standby;

[0077] S4: the viable cell count of the bacillus velezensis KN-901 spore powder obtained in S2 was adjusted to 200 billion CFU / g, and the mass percentage of the kasugamycin obtained in S2 was 6.1%, the mass percentage of the sodium lignosulfonate was 5.5%, the mass percentage of the polyformaldehyde naphthalene sulfonate sodium salt was 2.5%, and the mass percentage of the kaolin was 60%, and the above adjusted bacillus velezensis spore powder was fully stirred and mixed uniformly, and sieved to obtain a composite fungicide wettable powder.

[0078] Example 7: Application of the composite fungicide suspension agent in the prevention and treatment of potato scab

[0079] In this example, the land where potato scab occurred last year was selected as the test field, and three times of drip irrigation were carried out at the potato budding stage, the flowering stage and the tuber formation stage. The sample was the composite fungicide suspension agent obtained by the method in Example 5, and the conventional treatment was used as a blank control group. The dosing regimen is shown in Table 5.

[0080] The potato scab disease grading standard is shown in Table 6. Five-point sampling method was used to investigate and record at the potato harvesting stage. According to the above grading standard, the incidence of potato scab was graded, and the incidence rate, disease index, control effect of potato scab in each area, and the yield of crops in each treatment area, and the yield increase rate relative to the blank control group were calculated.

[0081]

[0082] Table 5

[0083] Group Treatment regimen Control group Routine treatment Treatment group 1 60ml / acre Bacillus velezensis KN-901 fermentation liquor Treatment group 2 60ml / acre 3% kasugamycin Treatment group 3 60ml / acre 3% kasugamycin compounded with Bacillus velezensis KN-901 suspension concentrate Treatment group 4 120ml / acre 3% kasugamycin compounded with Bacillus velezensis KN-901 suspension concentrate

[0084] Table 6

[0085] Classification Classification criteria Grade 0 No disease spots on the tuber Grade 1 Scab spot area accounts for 0-5% of the entire tuber area Grade 2 Scab spot area accounts for 5-10% of the entire tuber area Grade 3 Scab spot area accounts for 10-25% of the entire tuber area Grade 4 Scab spot area accounts for 25-50% of the entire tuber area Grade 5 Scab spot area accounts for more than 50% of the entire tuber area

[0086] As shown in Table 7, compared with the control group and the treatment group 1-2, the treatment group 3-4 using the composite fungicide suspension agent provided by the present application reduced the incidence of potato scab in the field to 31.74%-42.56% and the disease index to 9.31-15.16. Among them, the control effect of the 60ml / acre dosage can reach 65.33%, and with the increase of the dosage of the composite fungicide suspension agent, the control effect of the composite fungicide suspension agent can be improved to 78.71%. Therefore, the composite fungicide suspension agent provided by the present application can effectively reduce the incidence and disease index of potato scab, and has good potato scab control effect.

[0087] Table 7

[0088] Group Incidence rate (%) Disease index Control effect (%) Control group 87.26 43.73 - Treatment group 1 55.63 23.59 46.06 Treatment group 2 68.14 28.78 34.19 Treatment group 3 42.56 15.16 65.33 Treatment group 4 31.74 9.31 78.71

[0089] As shown in Table 8, the yield of field crops of treatment groups 3-4 using the composite fungicide suspension agent provided by the present application is increased to 3259 kg and 3512 kg respectively compared with the control group and treatment groups 1-2; the yield of field crops is increased by 13.63% when the use amount of the composite fungicide suspension agent is 60 ml / acre, and the yield of field crops is increased by 22.45% when the use amount is 120 ml / acre. It can be seen that the composite fungicide suspension agent provided by the present application has a certain growth-promoting effect on field crops, and the growth-promoting effect is significantly higher than the yield-increasing effect of the single dose of Bacillus velezensis KN-901 and kasugamycin.

[0090] Table 8

[0091]

[0092]

[0093] Example 8: Application of the composite fungicide wettable powder in preventing and treating yam anthracnose

[0094] In this example, the land with yam anthracnose disease occurring continuously for two years is selected as the test field, and the composite fungicide wettable powder obtained in Example 7 is sprayed once at the early stage of yam anthracnose disease, and the second spraying is performed 15 days after the use of the drug. The conventional treatment is used as a blank control group.

[0095] The yam anthracnose disease prevention and treatment drug scheme is shown in Table 9, the yam anthracnose disease grading standard is shown in Table 10, the 5-point sampling method is used to investigate and record at the high incidence period of yam anthracnose disease, 10 plants are collected as samples at each point, the disease condition of yam plants is graded according to the above grading standard, and the disease index and control effect of yam anthracnose disease in each treatment area are counted, and the yield of crops in each treatment area and the yield increase rate relative to the blank control group are investigated at the harvest period.

[0096]

[0097] Table 9

[0098] Group Treatment regimen Control group Routine treatment Treatment group 1 40g / acre Bacillus velezensis KN-901 powder Treatment group 2 40ml / acre 6% kasugamycin Treatment group 3 40g / acre 6% kasugamycin compounded with Bacillus velezensis KN-901 wettable powder Treatment group 4 80g / acre 6% kasugamycin compounded with Bacillus velezensis KN-901 wettable powder

[0099] Table 10

[0100] Classification Classification criteria Grade 0 No disease spots on the leaf surface Grade 1 Leaf disease spot area accounts for 0-5% of the total leaf area Grade 2 Leaf disease spot area accounts for 5-10% of the total leaf area Grade 3 Leaf disease spot area accounts for 10-30% of the total leaf area Grade 4 Leaf disease spot area accounts for 30-50% of the total leaf area Grade 5 Leaf disease spot area accounts for more than 50% of the total leaf area

[0101] As shown in Table 11, the treatment groups 3-4 using the composite fungicide wettable powder provided by the present application can reduce the disease index of yam anthracnose in the field to 7.45-11.23, relative to the control group and the treatment groups 1-2; wherein the control effect of the 40 g / acre use dose can reach 71.02%, and the control effect increases up to 80.77% with the increase of the use amount of the composite fungicide wettable powder. It can be seen that the composite fungicide wettable powder provided by the present application can effectively reduce the disease index of yam anthracnose, and has good yam anthracnose control effect.

[0102] Table 11

[0103] Group Disease index Control effect (%) Control group 38.75 - Treatment group 1 18.49 52.28 Treatment group 2 22.91 40.88 Treatment group 3 11.23 71.02 Treatment group 4 7.45 80.77

[0104] As shown in Table 12, the treatment groups 3-4 using the composite fungicide wettable powder provided by the present application can increase the yield of field crops to 1624 kg and 1796 kg, respectively, relative to the control group and the treatment groups 1-2, the yield increase rate of the 40 g / acre use dose is 17.17%, and the yield increase rate of the composite fungicide wettable powder is 29.58% when the use amount is increased to 80 g / acre. It can be seen that the composite fungicide wettable powder provided by the present application has a certain growth-promoting effect on field crops, which is significantly higher than the yield increase effect of the single dose of Bacillus velezensis KN-901 and kasugamycin.

[0105] Table 12

[0106] Group 667m 2 (kg)]]> Yield increase rate (%) Control group 1386 - Treatment group 1 1505 8.59 Treatment group 2 1401 1.08 Treatment group 3 1624 17.17 Treatment group 4 1796 29.58

[0107] Example 9: Application of the composite fungicide suspension in the prevention and treatment of sugar beet root rot

[0108] In this example, the land where the sugar beet root rot disease occurred last year is selected as the test field, and the composite fungicide suspension obtained by the method in Example 6 is drip irrigated on the test field 10 days after sowing, and a secondary root irrigation treatment is carried out during the tuber formation period. The conventional treatment is used as a blank control group.

[0109] The sugar beet root rot control scheme is shown in Table 13, the sugar beet root rot disease grading standard is shown in Table 14, the 5-point sampling method is used to investigate and record during the high incidence period of sugar beet root rot, the disease condition of the sugar beet plants is graded according to the above grading standard, and the disease index, control effect, crop yield and sugar content of each treatment area, and the yield increase rate and sugar content increase rate relative to the blank control group are calculated.

[0110]

[0111] Table 13

[0112] Group Treatment regimen Control group Routine treatment Treatment group 1 60ml / acre Bacillus velezensis KN-901 fermentation liquor Treatment group 2 60ml / acre 3% kasugamycin Treatment group 3 60ml / acre 3% kasugamycin compounded with Bacillus velezensis KN-901 suspension concentrate Treatment group 4 120ml / acre 3% kasugamycin compounded with Bacillus velezensis KN-901 suspension concentrate

[0113] Table 14

[0114] Classification Classification criteria Grade 0 Normal root growth without disease spots Grade 1 Mild disease of root tissue surface, with 0-10% of rot area Grade 2 Root rot spreads to the inside, with 10-40% of rot area Grade 3 Root rot spreads to the inside, with 40-50% of rot area Grade 4 Root rot spreads to the inside, with 50-70% of rot area Grade 5 Root rot spreads to the inside, with more than 70% of rot area

[0115] As shown in Table 15, the treatment groups 3-4 using the composite fungicide suspension provided by the present application reduced the disease index of sugar beet root rot to 5.43-7.63 relative to the control group and the treatment groups 1-2; wherein the control effect of the use dose of 60 ml / acre can reach 64.64%, and when the use amount of the composite fungicide suspension is increased to 120 ml / acre, the control effect is as high as 74.84%. It can be seen that the composite fungicide suspension provided by the present application can effectively reduce the disease index of sugar beet root rot, and has good effect of preventing and treating sugar beet root rot.

[0116] Table 15

[0117] Group Disease index Control effect (%) Control group 21.58 - Treatment group 1 12.67 41.29 Treatment group 2 18.69 13.39 Treatment group 3 7.63 64.64 Treatment group 4 5.43 74.84

[0118] As shown in Table 16, the yields of the treatment groups 3-4 using the composite fungicide suspension provided by the present application in the field were 10241 kg and 11058 kg, respectively, and the yield increase rates were 30.39% and 40.79%, respectively, when the use amount of the composite fungicide suspension was 60 ml / acre and 120 ml / acre compared with the control group; the sugar contents of the treatment groups 3-4 using the composite fungicide suspension provided by the present application in the field were 18.18% and 19.88%, respectively, and the sugar content increase rates were 16.24% and 27.11%, respectively, when the use amount of the composite fungicide suspension was 60 ml / acre and 120 ml / acre compared with the control group. It can be seen that the composite fungicide suspension provided by the present application has a certain growth-promoting effect on field crops, which is significantly higher than the yield-increasing effect of the single dose of Bacillus velezensis KN-901 and kasugamycin.

[0119] Table 16

[0120] Group 667m 2 Root yield (kg) Yield increase rate (%) Sugar content (%) Increase rate (%) Control group 7854 - 15.64 - Treatment group 1 9743 24.05 17.34 10.87 Treatment group 2 8231 4.80 16.14 3.20 Treatment group 3 10241 30.39 18.18 16.24 Treatment group 4 11058 40.79 19.88 27.11

[0121] Example 10: Application of composite fungicide wettable powder in preventing and treating rice blast

[0122] In this example, the land with rice blast disease occurring continuously for two years was selected as the test field, and the composite fungicide wettable powder obtained by the method disclosed in Example 7 was sprayed once after being mixed with water at the initial stage of rice blast, and the second spraying was performed again 10 days after the drug administration, and the conventional treatment was used as a blank control group.

[0123] The rice blast disease prevention and treatment drug regimen is shown in Table 17, the rice blast disease condition grading standard is shown in Table 18, the 5-point sampling method is used to investigate and record in the high incidence period of rice blast disease, 10 clusters of rice are collected as samples per point, the disease condition of rice is graded according to the above grading standard, and the disease index, prevention and treatment effect of rice blast disease in each treatment area, and the yield of crops in each treatment area and the yield increase rate relative to the blank control group are counted.

[0124]

[0125] Table 17

[0126] Group Treatment regimen Control group Routine treatment Treatment group 1 40g / acre Bacillus velezensis KN-901 powder Treatment group 2 40ml / acre 6% kasugamycin Treatment group 3 40g / acre 6% kasugamycin compounded with Bacillus velezensis KN-901 wettable powder Treatment group 4 80g / acre 6% kasugamycin and bacillus velezensis KN-901 wettable powder

[0127] Table 18

[0128]

[0129]

[0130] As shown in Table 19, compared with the control group and the treatment groups 1-2, the treatment groups 3-4 using the composite fungicide wettable powder provided by the present application can reduce the disease index of rice blast in the field to 2.49-4.06; wherein the prevention and treatment effect of the 40g / acre use dose can reach 82.85%, and the prevention and treatment effect is increased by up to 89.48% with the increase of the use amount of the composite fungicide wettable powder. It can be seen that the composite fungicide wettable powder provided by the present application can effectively reduce the disease index of rice blast, and has good rice blast prevention and treatment effect.

[0131] Table 19

[0132] Group Disease index Control effect (%) Control group 23.67 - Treatment group 1 9.04 67.81 Treatment group 2 6.34 73.22 Treatment group 3 3.58 84.88％ Treatment group 4 2.08 91.21％

[0133] As shown in Table 20, the treatment groups 3-4 using the composite fungicide wettable powder provided by the present application increase the yield of crops in the field to 491kg and 518kg respectively compared with the control group and the treatment groups 1-2, the yield increase rate of the 40g / acre use dose is 37.92%, and the yield increase rate of the composite fungicide wettable powder is increased to 80g / acre is 45.51%. It can be seen that the composite fungicide wettable powder provided by the present application has a certain growth-promoting effect on crops in the field, which is significantly higher than the yield increase effect of Bacillus velezensis KN-901 and spring mycin single agent.

[0134] Table 20

[0135] Group 667m 2 yield (kg)]] Yield increase rate (%) Control group 431 / Treatment group 1 510 18.33 Treatment group 2 538 24.83 Treatment group 3 601 39.44 Treatment group 4 629 45.94

[0136] Example 11: Application of composite fungicide suspension in prevention and treatment of bacterial fruit spot of Hami melon

[0137] The embodiment selects the land where bacterial fruit spot disease of Hami melon occurred last year as the test field, and sprays the composite fungicide suspension obtained by the method in Example 6 at the early stage of the disease, and sprays again 10 days after the spraying. The conventional treatment is used as a blank control group.

[0138] The spraying scheme for preventing bacterial fruit spot disease of Hami melon is shown in Table 21, the disease grading standard of bacterial fruit spot disease of Hami melon is shown in Table 22, the 5-point sampling method is used to investigate and record at the high incidence period of bacterial fruit spot disease of Hami melon, the disease condition of Hami melon plants is graded according to the above grading standard, and the disease index and control effect of bacterial fruit spot disease of Hami melon in each area are counted. The yield of Hami melon in each treatment area is determined at the harvest period, and the yield increase rate relative to the blank control group is determined.

[0139]

[0140] Table 21

[0141] Group Dosage regimen Control group Conventional treatment Treatment group 1 60ml / acre bacillus velezensis KN-901 fermentation liquor Treatment group 2 60ml / acre 3% kasugamycin Treatment group 3 60ml / acre 3% kasugamycin and bacillus velezensis KN-901 suspension concentrate Treatment group 4 120ml / acre 3% kasugamycin and bacillus velezensis KN-901 suspension concentrate

[0142] Table 22

[0143] Classification Classification standard 0 level No lesion 1 level Lesion area accounts for less than 6% of the whole leaf area 2 level Lesion area accounts for 6-10% of the whole leaf area 3 level Lesion area accounts for 11-20% of the whole leaf area 4 level Lesion area accounts for 21-50% of the whole leaf area 5 level Lesion area accounts for more than 50% of the whole leaf area

[0144] As shown in Table 23, compared with the control group and treatment groups 1-2, the treatment groups 3-4 using the composite fungicide suspension provided by the present application reduce the disease index of Hami melon in the field to 5.81-7.69; wherein the control effect of the use amount of 60 ml / acre can reach 75.63%, and when the use amount of the composite fungicide suspension is increased to 120 ml / acre, the control effect is as high as 81.59%. It can be seen that the composite fungicide suspension provided by the present application can effectively reduce the disease index of bacterial fruit spot disease of Hami melon, and has good effect on preventing bacterial fruit spot disease of Hami melon.

[0145] Table 23

[0146]

[0147]

[0148] As shown in Table 24, the yields of the treatment groups 3-4 using the composite fungicide suspension provided by the present application in the field are 2476 kg and 2613 kg, respectively, and when the use amount of the composite fungicide suspension is 60 ml / acre and 120 ml / acre, the yield increase rates relative to the control group are 29.23% and 36.38%, respectively. It can be seen that the composite fungicide suspension provided by the present application has a certain growth-promoting effect on the field crops, which is significantly higher than the yield increase effect of Bacillus velezensis KN-901 and spring mycin single agent.

[0149] Table 24

[0150] Group 667m 2 Root yield (kg) Yield increase rate (%) Control group 1916 - Treatment group 1 2158 12.63 Treatment group 2 2099 9.55 Treatment group 3 2476 29.23 Treatment group 4 2613 36.38

[0151] The content not described in detail in the specification of the present application is known to those skilled in the art. Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the scope of protection of the present application should be defined by the claims.

Claims

1. A type of Bacillus belye KN-901, characterized in that, The Bacillus belyssus KN-901 has the accession number CCTCC M 20242442 and is classified as follows: Bacillus velezensis Bacillus belye, deposited at the China Center for Type Culture Collection on November 6, 2024.

2. The Bacillus belyssus KN-901 according to claim 1, characterized in that, The 16S rRNA sequence of the Bacillus belyssus KN-901 is shown in SEQ ID NO.

1.

3. The use of Bacillus belye KN-901 as described in any one of claims 1 to 2 in the preparation of plant fungicides.

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