Bacillus velezensis and use thereof

Abstract

Provided are a strain of Bacillus velezensis EA19 and the use thereof. The strain is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO:M2023602. The strain has a relatively good treatment effect and prevention effect on stripe rust of Triticuma aestivum, and shows a comparable control efficacy to triadimefon. Moreover, the strain has an effect of reducing dosage while enhancing efficacy when used in combination with mefentrifluconazole. Therefore, the strain has important application values in the control of agricultural pests and diseases, provides a new way for green control of stripe rust of Triticuma aestivum, and has broad application prospects.

Description

Bacillus velez and its application Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to Bacillus velezensis and application thereof in the field of biological control. Background Art

[0002] Wheat (Triticuma aestivum) is one of the four major crops essential for human survival. Over one-third of the world's population relies on wheat as their staple food, and its yield and quality directly impact human survival and quality of life. With changes in farming systems, fertilizer and water conditions, and climate, wheat diseases have become a major constraint to high and stable wheat yields. In recent years, major wheat diseases occurring in my country include powdery mildew, fusarium head blight, sheath blight, stripe rust, root rot, and yellow dwarf disease.

[0003] Wheat stripe rust is an important airborne fungal disease of leaves caused by infection with Puccinia striiformis f.sp.Tririci. The pathogen can be spread over long distances by air currents and repeatedly infect wheat in the same growing season, causing damage.

[0004] Wheat stripe rust is a widespread, airborne disease that has the most severe impact on wheat production in my country. In recent years, due to climate change and shifts in cropping patterns, stripe rust has become more frequent, causing increased losses and posing a significant threat to wheat production. Because the wheat stripe rust fungus and wheat share a classic gene-for-gene relationship, cultivar resistance plays a crucial role in the prevention and control of stripe rust. However, due to the overlapping generations of sexual and asexual reproduction in nature, wheat stripe rust has numerous, low-toxic strains and rapid mutation rates. Varieties containing a single resistance gene are often overcome by toxic variations in the stripe rust, rendering them unusable in production. Therefore, current control of this disease relies primarily on chemical agents.

[0005] Triazole pesticides, represented by triadimefon and tebuconazole, have long been the preferred agents for controlling stripe rust. As of December 20, 2022, of the 32 registered active ingredients for wheat stripe rust, 12 were triazoles, accounting for 37.5%; 175 pesticide varieties, or 82.5%, contained triazoles. Due to the single, long-term application of triazole pesticides, resistance has emerged in production, resulting in reduced field control effectiveness. Furthermore, with the promotion and development of green plant protection concepts in recent years, green control has attracted increasing attention. Emerging green control products such as microbial pesticides, immune elicitors, and RNAi pesticides are also becoming research hotspots. Therefore, screening novel green control products can provide reliable technical solutions for green control of wheat stripe rust.

[0006] So far, there is no report on using Bacillus velezensis for preventing and treating wheat stripe rust. SUMMARY

[0007] In view of the deficiencies in the prior art, the purpose of the present application is to provide a new Bacillus velezensis strain which can effectively prevent and treat wheat stripe rust, wheat sharp eyespot and wheat scab. The Bacillus velezensis strain of the present application also has the characteristics of high environmental friendliness. When used in combination with chemical pesticides, the amount of chemical pesticides can be reduced, while the prevention and treatment effect remains basically unchanged, or even higher.

[0008] According to a first aspect of the present application, a Bacillus velezensis strain is provided, which is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M2023602 and a preservation date of April 24, 2023.

[0009] In some embodiments, the 16S rRNA sequence of the strain has a nucleotide sequence as shown in SEQ ID NO: 1.

[0010] In some embodiments, the Bacillus velezensis strain of the present disclosure is a gram-positive bacterium. In some embodiments, the Bacillus velezensis strain of the present disclosure can produce spores.

[0011] In some embodiments, the arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease and / or tryptophanase of the Bacillus velezensis strain of the present disclosure are negative for enzyme activity.

[0012] In some embodiments, the gelatinase activity of the Bacillus velezensis strain of the present disclosure is positive.

[0013] In some embodiments, the Bacillus velezensis strain of the present disclosure is negative for enzyme activity related to citrate, sodium thiosulfate or tryptophan.

[0014] In some embodiments, the Bacillus velezensis strain of the present disclosure is positive for enzyme activity related to pyruvate.

[0015] In some embodiments, the Bacillus velezensis strain of the present disclosure is negative or weakly positive for fermentation or oxidation results of glucose, mannitol, myo-inositol, sorbitol, rhamnose, sucrose, melibiose, and / or amygdalin. In some embodiments, the Bacillus velezensis strain of the present disclosure is negative for fermentation or oxidation results of glucose, mannitol, myo-inositol, rhamnose, and / or amygdalin. In some embodiments, the Bacillus velezensis strain of the present disclosure is weakly positive for fermentation or oxidation results of sorbitol, sucrose, and / or melibiose.

[0016] In some embodiments, the Bacillus velezensis strain of the present disclosure is positive for fermentation or oxidation results of arabinose.

[0017] In some embodiments, the Bacillus velezensis strain of the present disclosure is capable of utilizing one or more of arabinose, ribose, xylose, glucose, fructose, mannose, myo-inositol, mannitol, sorbitol, methyl glucoside, arbutin, esculin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, raffinose, starch, and glycogen.

[0018] In some embodiments, the Bacillus velezensis strain of the present disclosure is unable to hydrolyze, degrade, or decompose arginine, lysine, ornithine, urea, and / or tryptophan. In some embodiments, the Bacillus velezensis strain of the present disclosure is unable to utilize sodium citrate. In some embodiments, the Bacillus velezensis strain of the present disclosure is unable to utilize sodium thiosulfate, produce H2S, urea, tryptophan, tryptamine. In some embodiments, the Bacillus velezensis strain of the present disclosure is unable to utilize tryptophan to produce indole. In some embodiments, the Bacillus velezensis strain of the present disclosure is capable of utilizing 3-hydroxybutanone to produce acetyl methyl carbinol. In some embodiments, the Bacillus velezensis strain of the present disclosure is capable of decomposing gelatin. In some embodiments, the Bacillus velezensis strain of the present disclosure is unable to ferment or oxidize glucose, mannitol, myo-inositol, rhamnose, and / or amygdalin. In some embodiments, the Bacillus velezensis strain of the present disclosure is capable of fermenting or oxidizing arabinose.

[0019] In some embodiments, the Bacillus velezensis strain of the present disclosure is capable of utilizing one or more of the following carbon sources: glycerol, arabinose, ribose, xylose, glucose, fructose, mannose, myo-inositol, mannitol, sorbitol, methyl glucoside, arbutin, esculin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, raffinose, starch, and glycogen. In some embodiments, the Bacillus velezensis strain of the present disclosure is capable of utilizing the above-mentioned carbon sources to produce acid.

[0020] According to a second aspect of the present application, there is provided use of the Bacillus velezensis strain of the present disclosure in the prevention of plant diseases.

[0021] In some embodiments, the plant disease can include a cereal (e.g., wheat or barley) disease. In some embodiments, the plant disease can include, but is not limited to, cereal powdery mildew, scab, sharp eyespot, rust (e.g., stripe rust, leaf rust, and stem rust), root rot, and yellow dwarf. In specific embodiments, the plant disease can include, but is not limited to, wheat stripe rust, scab, and sharp eyespot.

[0022] In some embodiments, the Bacillus velezensis strain of the present disclosure can have antagonistic effects on pathogenic fungi that cause the plant disease.

[0023] In some embodiments, the Bacillus velezensis strain of the present disclosure can have antagonistic effects on pathogenic fungi selected from the group consisting of:

[0024] In some embodiments, the pathogenic fungi can include, but is not limited to, Ceratobasidium graminearum (Bourd.), Rhizoctonia cereadis, Puccinia striiformis West. f. sp. tritici, P. triticina f. sp. tritici Eriks., P. graminis Pers. f. sp. tritici Eriks., F. graminearum, F. asiaticum, F. culmorum, F. avenaceum, Fusarium acuminatum (ElL et Ev) Wr, F. moniliforme, F. nivale, and the like.

[0025] In some embodiments, the Bacillus velezensis strain of the present disclosure can be in the form of granules, wettable powders, aqueous solutions, and the like.

[0026] In some embodiments, the Bacillus velezensis strain of the present disclosure can be used in combination with other fungicides to prevent plant diseases.

[0027] In some embodiments, the fungicide may include one or more of triazole fungicides, benzimidazole fungicides, imidazole fungicides, methoxyacrylate fungicides, cyanoacrylate fungicides, amide fungicides, organosulfur fungicides, and organochlorine fungicides.

[0028] In some embodiments, the fungicide may include tebuconazole, carbendazim, oxazolidinone, flutolanil, oxazolidinone, isopyram, fluopyram, fenfuran, carboxin, oxycarboxin, thiofuran, fluopyram, bifenazolin, isopyram, boscalid, oxepiconazole, bifenthionil, cyproconazole, difenoconazole, diniconazole, fluepiconazole, ethiconazole, nitrobenzene, fluquinconazole, flusilazole, flutriafol, hexaconazole, imipenem , one or more of iodide, chlorfluanidazole, metconazole, myclobutanil, penconazole, propiconazole, silyconazole, tebuconazole, tetrafluanidazole, triadimefon, triadimenol, trithiocarb, prothioconazole, azoxystrobin, syringosterone, enoxastrobin, flutoxin, picoxystrobin, pyraclostrobin, pyraclostrobin, pyraclostrobin, chlorpyrifos, kresoxim-methyl, trifloxystrobin, etherstrobin, enoxastrobin, oxadiazon, oxadiazon, flutoxin, and pyraclostrobin.

[0029] In some specific embodiments, the Bacillus velezensis strain disclosed herein can be used in combination with meclofentoxim-azole to control wheat diseases, such as wheat powdery mildew, fusarium head blight, sheath blight, stripe rust, root rot, and yellow dwarf disease.

[0030] According to a third aspect of the present invention, a composition is provided, comprising the Bacillus velezensis strain disclosed herein.

[0031] In some embodiments, the content of the spores of Bacillus velezinsis in the composition is ≥10.0×10 5 CFU / g.

[0032] In some embodiments, the composition comprises more preferably ≥10.0×10 10 CFU / gram of the Bacillus velezensis strain.

[0033] In some embodiments, the composition comprises at least 50% or more of the live spores of Bacillus velezensis, more preferably including 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the live spores of Bacillus velezensis.

[0034] In some embodiments, the composition can further include a fungicide. In some embodiments, the fungicide can include one or more of a triazole fungicide, a benzimidazole fungicide, an imidazole fungicide, a strobilurin fungicide, a cyanoacrylate fungicide, an amide fungicide, an organosulfur fungicide, an organochlorine fungicide.

[0035] In some embodiments, the fungicide can include one or more of tebuconazole, carbendazim, carpropamid, fluazinam, iprodione, isopropothioamide, fluopyram, fenfuram, carboxin, oxycarboxin, thifluzamide, pyraclostrobin, bixafen, isopropothioamide, boscalid, cyprodinil, bitertanol, fenpropimorph, cyproconazole, difenoconazole, fenpropidin, cyprodinil, prothioconazole, fluquinconazole, ethaboxam, silthiofam, prothioconazole, metconazole, triclozole, uniconazole, myclobutanil, cyproconazole, simeconazole, prothioconazole, tebuconazole, tetraconazole, triflumizole, uniconazole, ipconazole, propiconazole, silthiofam, tebuconazole, tetraconazole, triflumizole, uniconazole, myclobutanil, prothioconazole, simeconazole, iprodione, boscalid, prothioconazole, cyproconazole, simeconazole, and pyraclostrobin.

[0036] In some embodiments, the weight ratio of the Bacillus velezensis to the fungicide in the composition can be (1-10): 1. In some embodiments, the weight ratio of the Bacillus velezensis to the fungicide in the composition can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0037] In some embodiments, the composition can be in the form of a powder, a microencapsulated powder, a capsule, a tablet, a lozenge, a granule, an emulsion, a suspension, a spray formulation, an electrostatic spray formulation, a suppository, or a wettable powder.

[0038] According to a fourth aspect of the present application, there is provided use of the composition of the present disclosure in the control of wheat diseases. In some embodiments, the wheat diseases include wheat stripe rust, wheat scab, and wheat sharp eyespot.

[0039] According to a fifth aspect of the present application, there is provided a bacterial agent comprising the Bacillus velezensis strain of the present disclosure.

[0040] In some embodiments, the bacterial agent can be in the form of granules, wettable powder or water agent.

[0041] In some embodiments, the bacterial agent can be in the form of wettable powder. In some embodiments, the bacterial agent can comprise, by weight parts, 5-20% of Bacillus velezensis strain raw powder, 0.5-2% of dispersant, 0.5-2% of wetting agent, and the rest of filler.

[0042] In some embodiments, the bacterial agent can comprise, by weight parts, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 18% or 20% of Bacillus velezensis strain raw powder.

[0043] In some embodiments, the bacterial agent can comprise, by weight parts, 0.5%, 1.0%, 1.5% or 2% of dispersant.

[0044] In some embodiments, the bacterial agent can comprise, by weight parts, 0.5%, 1.0%, 1.5% or 2% of wetting agent.

[0045] In some embodiments, the dispersant can be at least one of sodium lignosulfonate, calcium lignosulfonate, naphthalene sulfonate formaldehyde condensate and carboxylate salt polymer. In some embodiments, the dispersant can be naphthalene sulfonate formaldehyde condensate.

[0046] In some embodiments, the wetting agent can be at least one of sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl laurate and alkyl naphthalene sulfonate. In some embodiments, the wetting agent can be alkyl naphthalene sulfonate.

[0047] According to a sixth aspect of the present application, there is provided a method for preparing the bacterial agent described above, the method comprising the steps of: obtaining a fermentation broth containing spores of the Bacillus velezensis strain; performing acidification treatment; filtering to obtain a filtrate, adding a stabilizer to the filtrate, and then performing spray drying to obtain strain raw powder; and mixing the strain raw powder with a dispersant, a wetting agent and a filler to obtain the bacterial agent of the Bacillus velezensis strain.

[0048] In some embodiments, the stabilizer can be selected from the group consisting of trehalose, dextran, sucrose.

[0049] In some embodiments, the acidification treatment adjusts the pH of the fermentation broth to 3.0-5.0, for example, to 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0050] In some embodiments, the dispersing agent can be at least one of sodium lignosulfonate, calcium lignosulfonate, naphthalene sulfonate formaldehyde condensate, and carboxylate salt polymer. In some embodiments, the dispersing agent can be naphthalene sulfonate formaldehyde condensate.

[0051] In some embodiments, the wetting agent can be at least one of sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl laurate, and alkyl naphthalene sulfonate. In some embodiments, the wetting agent can be alkyl naphthalene sulfonate.

[0052] The present disclosure isolates a new Bacillus velezensis strain from fresh wheat plants in the original seed field in Xiangyang City, Hubei Province. And through experiments, it is verified to have the following beneficial effects:

[0053] 1) It has good treatment and prevention effects on wheat stripe rust.

[0054] 2) When used to prevent and treat wheat stripe rust, it can reduce the amount of chemical pesticide, chlorothalonil, without reducing the prevention and treatment effect, and relieve the resistance of wheat and other crops to chlorothalonil.

[0055] 3) The significant effect of the Bacillus velezensis strain of the present disclosure in preventing and treating wheat stripe rust makes the strain have important application value in the prevention and treatment of agricultural pests and diseases, provides a new way for green prevention and treatment of wheat stripe rust, and has broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 is a Neighbor-Joining phylogenetic tree of the Bacillus velezensis strain EA19 of the present disclosure based on 16S rRNA gene sequences.

[0057] Figure 2 is a photo of the colony morphology and bacterial morphology under a microscope of the Bacillus velezensis strain EA19 of the present disclosure. Wherein, A is a photo of the bacterial morphology of the Bacillus velezensis strain EA19 under a microscope (1000X), and B is the colony morphology of the Bacillus velezensis strain EA19 on LB medium.

[0058] Figure 3 is the pot active test results of Bacillus velezensis strain EA19 against Puccinia striiformis f. sp. tritici, wherein a is water control, b is EA19 treatment, c is 15% triadimefon treatment.

[0059] Figure 4 is the indoor control effect of Bacillus velezensis strain EA19 mixed with chlorothalonil against Puccinia striiformis f. sp. tritici, wherein a is water control, b is EA19 treatment, c is 400 g / L chlorothalonil suspension treatment, d is EA19 + chlorothalonil suspension treatment.

[0060] Figure 5 is the field control effect of Bacillus velezensis strain EA19 mixed with chlorothalonil against Puccinia striiformis f. sp. tritici, from left to right, water control, chlorothalonil suspension (25 g / acre), EA19 wettable powder (100 g / acre), EA19 (100 g / acre) + chlorothalonil suspension (20 g / acre), chlorothalonil suspension (20 g / acre). DETAILED DESCRIPTION

[0061] In order to make the objectives, technical solutions, and advantages of the present application clearer, the following further describes the present application in conjunction with embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessarily obscuring the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0062] The following are specific embodiments of the present application, which further describe the technical solutions of the present application, but the protection scope of the present application is not limited to these embodiments. Any changes or equivalent replacements that do not deviate from the concept of the present application are included in the protection scope of the present application.

[0063] EMBODIMENTS

[0064] Example 1. Isolation and identification of strain

[0065] 1) Isolation and purification of strain EA19

[0066] Fresh wheat roots, stems, and leaves collected from a seed field in Xiangyang City, Hubei Province were washed clean with tap water, cut into small pieces of 1 cm 2 around, soaked in 5% available chlorine sodium hypochlorite solution for 5 min, then soaked in 70% ethanol for 30 s, rinsed with sterile water 4 times, added with 1 mL sterile water and ground finely, and the juice was diluted to 10 4 , 10 5 , and 10 6The 200 μL was coated on a nutrient agar NA (3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 20 g of agar powder per 1000 mL, pH 7.0) plate. After 48 h of culture at 30°C, single colonies were picked and isolated by streaking multiple times. The isolated strain was stored at -70°C and named as strain EA19.

[0067] 2) Molecular identification of strain EA19

[0068] The 16S rRNA of strain EA19 was sequenced by Wuhan Tianyi Huayu Gene Technology Co., Ltd. The sequence of 16S rRNA of the strain is shown as SEQ ID NO: 1, and the sequence of gyrA gene is shown as SEQ ID NO: 2. The sequence was compared with the 16S rRNA sequence of Bacillus velezensis registered in Genbank, and the homology reached more than 99%.

[0069] Meanwhile, the Neighbor-Joining phylogenetic tree of strain EA19 was constructed with Alicyclobacillus acidocaldarius ATCC 27009 (AB042056) as the outgroup, as shown in Figure 1.

[0070] 3) Morphological characteristics identification of strain EA19

[0071] After 48 h of shaking culture of strain EA19 in NA liquid medium, staining was performed by methylene blue staining, Gram staining (crystal violet acid amine staining), and spore staining. It was observed that the colony was opaque, about 3-7 mm in size, with irregular edges, rough and granular surface like frosted glass or wax-like, no pigment secretion, Gram-positive bacteria, rod-shaped bacteria under a microscope, (1.0-1.3) x (3.0-5.0) μm, chain formation, motility, oval spores, endogenous or subterminal, and no obvious spore swelling.

[0072] Under a microscope (1000 times), and LB (10 g of peptone, 5 g of sodium chloride, 10 g of glucose, 10 g of yeast extract, 20 g of agar per liter of medium, and water to make up 1 liter) plate, the colony growth morphology was shown in Figure 2A and Figure 2B, respectively.

[0073] 4) Physiological and biochemical characteristics identification of strain EA19

[0074] After 2 Systems Version: 03.01 full-automatic microbial identification system analysis, the enzyme activity, carbon source oxidation and carbon source acid production results of strain EA19 were obtained, as shown in Table 1 and Table 2.

[0075] Table 1 Enzymatic activity, carbon source oxidation results of strain EA19 Note: + represents a positive reaction; - represents a negative reaction; W represents a weak positive reaction

[0076] Table 2 Acid production using carbon sources of strain EA19 +: positive reaction; - : negative reaction;

[0077] According to the cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence experimental data of the strain, and referring to the "Berger's Systematic Bacteriology Manual", the strain is identified as Bacillus velezensis. And based on the fact that the strain has different physiological and biochemical characteristics from existing Bacillus velezensis, it is a new strain of Bacillus velezensis. The strain is preserved in the China Center for Type Culture Collection (Wuhan, China), and the preservation number is CCTCC NO: M2023602, and the preservation date is April 24, 2023.

[0078] Example 2. Culture of Bacillus velezensis EA19

[0079] Step one, the strain Bacillus velezensis EA19 was inoculated in the slope culture medium prepared by LB culture medium to prepare the first grade slope seed, and cultured in a constant temperature incubator at 30±0.5℃ for 24h.

[0080] Step two, the slope seed in step one was inoculated in a flask or fermentation tank containing LB liquid medium (containing 10g of protein peptone, 5g of sodium chloride, 10g of glucose, 10g of yeast extract, and water to make up 1 liter per liter of medium), and the volume concentration of the seed liquid in the LB liquid medium was 10%, and the seed liquid was cultured in the constant temperature incubator at a temperature of 29-31℃ and a constant speed of 160r / min for 8h to enter the logarithmic growth phase, and then discharged to obtain the liquid seed.

[0081] Wherein, LB liquid medium is basically same as the slant medium, only without agar. The liquid culture step can also use FSTB medium (FeSO4·7H2O 0.025 g / L, NH4NO3 0.3 g / L, KCl 7 g / L, CaCl2 2.8 g / L, NaCl 20 g / L, beef extract 5 g / L, peptone 10 g / L, pH value is 7.0-7.5, 4 ml / L glycerol), SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose), YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl) or peptone medium (peptone 20 g, beef extract 3 g, sodium chloride 5 g).

[0082] Step three, through the fermentation method described above, the fermentation broth with more than 50 billion spores is obtained, industrial hydrochloric acid is added for acidification, the pH value of the fermentation broth is adjusted to 4.2 (measured by pH electrode), and a 150-mesh vibrating screen is used for filtration to obtain EA-19 filtrate; 1% (w / w%) trehalose is added to the filtrate, and after uniform mixing, spray drying tower is used for powdering, and the outlet temperature is controlled at 85°C to obtain EA-19 raw powder, and the spore number of the raw powder is 150 billion.

[0083] Step four, EA-19 raw powder with more than 1500 billion spores is prepared by the above method; raw materials are added to a double-cone mixer, and the addition ratio of the raw materials is EA-19 raw powder: dispersant Morwet D-425 (sodium salt of alkyl naphthalene sulfonate condensate, produced by AkzoNobel China Co., Ltd.): wetting agent Morwet EFW (mixture of alkyl naphthalene sulfonate and anionic wetting agent, produced by AkzoNobel China Co., Ltd.): ammonium sulfate filler (Balin Petrochemical) = 13:1:1:85, and the ingredient ratio is by weight; after uniform mixing, 100 billion EA-19 wettable powder is obtained.

[0084] Example 3. Effect of bacillus velezensis EA19 on the prevention and treatment of wheat stripe rust

[0085] Using the product obtained in Example 2, tests for the treatment and protection effects on wheat stripe rust were carried out, as follows.

[0086] The treatment method of the therapeutic effect is as follows: take the uniform one-leaf one-heart stage wheat seedlings, use electronic fluorination liquid to spray the wheat stripe disease spore suspension, the inoculation concentration of the wheat stripe disease spore is 88 mg of stripe rust spores in 30 mL of electronic fluorination liquid FC40, shake and mix uniformly, and then uniformly spray on 30 pots of wheat seedlings with a manual spray pot. After inoculation, place in a humidity incubator at a temperature of 16°C and a light cycle of L16h:D8h for culture, after 1 day of culture, spray each treatment agent, the amount of each treatment agent is 10 ml, 3 times, and the blank control is not sprayed, and the positive control is fluquinconazole.

[0087] The treatment method of the protective effect is as follows: take the uniform one-leaf one-heart stage wheat seedlings, first spray the agent, and then place the wheat seedlings in a humidity incubator at a temperature of 16°C and a light cycle of L16h:D8h for culture, after 1 day of culture, inoculate the wheat stripe disease spore suspension, and the inoculation method is the same as that in the treatment of the therapeutic effect.

[0088] After the first leaf of the wheat uniformly develops (about 14 days are needed), the severity of the first leaf of the wheat in all treatments is investigated, the diseased leaves are classified according to the standard of GB / T17980.21-2000, and the disease index and the control effect are calculated, and the control effect calculation formula is as follows:

[0089] Control effect (%) = (disease index of the control area - disease index of the treatment area) * 100 / disease index of the control area

[0090] The test results are shown in Table 3, the protective and therapeutic effects of 100 billion viable spores / g of Bacillus velezensis EA19 (200 g / acre) are 63.77% and 61.14% respectively, and the protective and therapeutic effects of the control agent 15% triticonazole (120 g / acre) are 68.87% and 64.65% respectively. The results show that there is no significant difference between the protective effect of 100 billion viable spores / g of Bacillus velezensis EA19 and the control effect of the chemical agent 15% triticonazole, and there is a significant difference between the therapeutic effect and the control agent. The results show that Bacillus velezensis EA19 has a good control effect on wheat stripe rust (Figure 3).

[0091] Table 3 Control effects of different biological agents and chemical agents on wheat stripe rust

[0092] Example 4. Effect of Bacillus velezensis EA19 and chemical agents on synergistic control of wheat stripe rust (indoor pot culture)

[0093] The present example found that the combination of Bacillus velezensis EA19 and chlorothalonil reduced the dosage and achieved unexpected results. The test method refers to the protection treatment of Example 3, and the concentration of the agent is shown in Table 4. The application amount of each treatment is 10 ml, 3 times, and the blank control is not applied. The control effect calculation method is the same as Example 3, and the specific test results are shown in Table 4 and Figure 4.

[0094] Table 4 Effect of Bacillus velezensis EA19 and chlorothalonil on synergistic control of wheat stripe rust Note: There is a significant difference between groups marked with different letters in the above table.

[0095] From the above test, it can be seen that 10 billion viable spores / g of Bacillus velezensis EA19 wettable powder (100 g / acre) has a control effect of 47.16% on stripe rust, while the combination of 10 billion viable spores / g of Bacillus velezensis EA19 (100 g / acre) and 400 g / L of chlorothalonil suspension concentrate (20 g / acre) has a control effect of 87.86%, which is unexpectedly much higher than the control effect of the two alone. These results show that the combination of Bacillus velezensis EA19 and chemical pesticide chlorothalonil has a synergistic effect of reducing the amount of pesticide and increasing the effect.

[0096] Example 5. Field test of Bacillus velezensis EA19 and chlorothalonil for controlling wheat stripe rust

[0097] Wheat was sown on November 5, 2022, and harvested on May 13, 2023, with a sowing amount of 225 kg / hm 2 , mechanical striping, fertilization, ditching and management according to conventional production measures, and no other fungicides were used. Each treatment was set up 4 times, with an area of 20 square meters. The test site was set up in Jiangbei Farm, Jiangling County, Jingzhou City, Hubei Province, and the stripe rust susceptible variety Tianmin 198 was planted. The pesticide was applied at the early stage of wheat stripe rust, and the control effect calculation method was the same as Example 3. The specific test results are shown in Table 5 and Figure 5.

[0098] Table 5 Effect of Bacillus velezensis EA19 and chlorothalonil on synergistic (mixed use) control of wheat stripe rust Note: There is a significant difference between groups marked with different letters in the above table.

[0099] From the above experiment, it can be seen that the control effect of 400 g / L myclobutanil suspension concentrate (25 g / mu) on wheat stripe rust is 79.93%; the control effect of 100 billion living spores / g Bacillus velezensis EA19 (100 g / mu) combined with 400 g / L myclobutanil suspension concentrate (20 g / mu) is unexpectedly significantly higher than that when they are used alone, which realizes the control effect equivalent to that of 400 g / L myclobutanil suspension concentrate (25 g / mu) while reducing the amount of fungicides, which is consistent with the indoor pot experiment. It also shows that after Bacillus velezensis EA19 and chemical pesticide myclobutanil are mixed, they can play a synergistic role of reducing drugs and increasing efficiency.

[0100] Example 6. Field test of Bacillus velezensis EA19 for controlling wheat sharp eyespot

[0101] Wheat sharp eyespot, also known as sharp eyespot, is a disease caused by Geratobasidium cornigeru (Bourd.) Rogers and occurs on wheat. Wheat sharp eyespot mainly occurs on leaf sheaths and stems. This embodiment tests the control effect of Bacillus velezensis EA19 of the present disclosure on wheat sharp eyespot.

[0102] Wheat was sown on November 5, 2022, and harvested on May 13, 2023, with a sowing amount of 225 kg / hm 2 , mechanical strip sowing, fertilization, ditching and management were carried out according to the production routine, and no other fungicides were used. Each treatment was set with 4 repetitions, and the area of each plot was 20 square meters. The test site was set in Jiangbei Farm, Jiangling County, Jingzhou City, Hubei Province, and the planting variety was Xinnong 979. The test was set up with 3 treatments: (1) 30 g of EA19 (100 billion living spores / g) was mixed with 170 mL of sterile water to seed 10 kg of wheat; (2) 5 mL of 6% tebuconazole was mixed with 195 mL of sterile water to seed 10 kg of wheat (pesticide control); (3) 200 mL of sterile water was used to seed 10 kg of wheat (blank control). The seeds were coated in the room 1 day before sowing, and then dried for standby. The control effect was investigated at the jointing stage of wheat, and the calculation method of the control effect was the same as that in Example 3. The test results are shown in Table 6.

[0103] Table 6. Effect of Bacillus velezensis EA19 on wheat sharp eyespot

[0104] From the above experiment, it can be seen that 100 billion living spores / g Bacillus velezensis EA19 wettable powder has a control effect of 69.93% on wheat sharp eyespot when used for seed dressing; and the control effect of chemical pesticide 6% tebuconazole is 72.93%, which shows that the control effects of Bacillus velezensis EA19 and chemical seed dressing agent tebuconazole are similar.

[0105] Example 7. Field test of Bacillus velezensis EA19 for controlling wheat scab

[0106] Wheat scab, also known as head blight, is a disease that occurs on wheat and is usually caused by multiple Fusarium species. Fusarium species that can cause wheat scab include, for example, Fusarium graminearum Schw., Fusarium avenaceum (Fr.) Sacc., Fusarium moniliforme Sheld., Fusarium culmorum (W.G. Smith) Sacc, and Fusarium acuminatum (Ell et Ev) Wr. This example demonstrates the efficacy of the Bacillus velezensis EA19 of the present disclosure for controlling wheat scab.

[0107] Wheat was sown on November 5, 2022, and harvested on May 13, 2023, with a sowing rate of 225 kg / hm 2 , mechanical strip sowing, fertilization, ditching and management were carried out according to the production routine, and no other fungicides were used. Each treatment was set up with 4 replicates, and the plot area was 20 square meters. The test site was set up in Jiangbei Farm, Jiangling County, Jingzhou City, Hubei Province, and the planting variety was Xinong 979. The test was set up with 3 treatments: (1) 100 grams of EA19 (100 billion viable spores / gram); (2) 25% carbendazim wettable powder (100 grams / acre); (3) water. The first application of the pesticide was on April 15, 2023, and the second application was on April 22 (7 days later). The disease ear rate and disease grade were investigated before the dough stage. A 0.667 m 2 square frame was used for investigation, and 4 replicates were used for each plot. The number of diseased ears and disease grade in the frame were recorded separately. The disease ear area was graded as a percentage of the total ear area, and the number of diseased ears and total ears were recorded. The scab investigation and recording were carried out in accordance with NY / T 1464.15-2007. The method for determining the content of deoxynivalenol (DON) in wheat kernels: refer to GBT 23503-2009 "Determination of Deoxynivalenol in Food by Immunoaffinity Chromatography Purification and High Performance Liquid Chromatography".

[0108] Table 7 Effect of Bacillus velezensis EA19 on controlling wheat scab

[0109] The field experiment results shown in Table 7 show that after spraying Bacillus mojavensis EA19, the control effect is 72.58% compared with the group without spraying, which is equivalent to the control effect of the conventional pesticide carbendazim, and the DON toxin control effect of wheat is 85.31%. Therefore, after spraying Bacillus mojavensis EA19, the scab disease can be effectively controlled, the DON content in the wheat grains can be reduced, and the quality of the wheat can be improved.

[0110] The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the protection scope of the present application.

Claims

1. A Bacillus velezensis strain, which is deposited in China General Microbiological Culture Collection Center, and has a deposit number of CCTCC NO:M2023602 and a deposit date of April 24, 2023.

2. The strain according to claim 1, characterized in that, The 16S rRNA sequence of the strain has a nucleotide sequence as shown in SEQ ID NO:

1.

3. The strain according to claim 1, characterized in that, The Bacillus velezensis strain has one or more of the following properties: 1) the strain is a gram-positive bacterium; 2) the strain is negative for enzyme activities of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease and / or tryptophanase; 3) the strain is positive for gelatinase activity; 4) the strain is negative for activities of enzymes related to utilization of citric acid, sodium thiosulfate or tryptophan; 5) the strain is positive for activity of enzymes related to utilization of pyruvate; 6) the strain is negative or weakly positive for fermentation or oxidation results of glucose, mannitol, myo-inositol, sorbitol, rhamnose, sucrose, melibiose and / or amygdalin 7) the strain is negative for fermentation or oxidation results of glucose, mannitol, myo-inositol, rhamnose and / or amygdalin; 8) the strain is weakly positive for fermentation or oxidation results of sorbitol, sucrose and / or melibiose; 9) the strain is positive for fermentation or oxidation results of arabinose; 10) the strain is capable of utilizing one or more of arabinose, ribose, xylose, glucose, fructose, mannose, myo-inositol, mannitol, sorbitol, methyl glucoside, arbutin, esculin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, raffinose, starch and glycogen. 4.The Bacillus velezensis strain of any one of claims 1 to 3 for use in plant disease control.

5. Use according to claim 4, characterized in that, The plant comprises wheat or barley, preferably wheat; The disease preferably comprises rust, scab and sharp eyespot; and / or The Bacillus velezensis strain has antagonistic effect on one or more of the following pathogenic fungi: Fusarium spp., Puccinia, Geratobasidium cornigeru (Bourd.) Rogers, Preferably, the strain has antagonistic effect on one or more of the following pathogenic fungi: Ceratobasidium graminearum (Bourd.), Rhizoctonia cereadis, Puccinia striiformis West. f. sp. tritici, P. triticina Eriks., P. graminis Pers. f. sp. tritici Eriks., F. graminearum, F. asiaticum, F. culmorum, F. avenaceum, F. acuminatum (ElL et Ev) Wr, F. moniliforme, F. nivale.

6. Use according to claim 4, characterized in that, The strain can be prepared into the form of granules, wettable powder or aqueous fungicide.

7. Use according to claim 4, characterized in that, The strain is applied in combination with other fungicides, Preferably, the fungicide includes one or more of triazole fungicides, benzimidazole fungicides, imidazole fungicides, methoxy acrylate fungicides, cyano acrylate fungicides, amide fungicides, organic sulfur fungicides, organic chlorine fungicides, More preferably, the fungicide includes one or more of tebuconazole, carbendazim, carpropamid, fluazinam, iprodione, isopropothioamide, fluopyram, fenfuram, carboxin, oxycarboxin, thifluzamide, pyraclostrobin, bixafen, isopropothioamide, boscalid, cyprodinil, bitertanol, fenpropidin, cyproconazole, difenoconazole, fenpropimorph, cyproconazole, cyprodinil, fluquinconazole, etridiazole, flusilazole, spiroxamin, hexaconazole, iminoctad, ipconazole, metconazole, tecloftalam, myclobutanil, pefurazoate, prochloraz, propiconazole, silthiofam, tebuconazole, tetraconazole, triflumizole, triflumizole, thiabendazole, prothioconazole, azoxystrobin, coumoxystrobin, enoxastrobin, fluoroimide, picoxystrobin, pyraclostrobin, pyribencarb, chlorfenazole, dimethomorph, enoxastrobin, dimoxystrobin, fenamidone, fluoxastrobin, metominostrobin, myclobutanil, and pyracarbolid, Preferably, the weight ratio of the Bacillus velezensis to the fungicide is (1-10):

1.

8. A composition comprising the Bacillus velezensis strain of any one of claims 1 to 3.

9. The composition of claim 8, wherein, The composition comprises > 10.0 x 10 5 CFU / gram of the strain, more preferably > 10.0 x 10 10 CFU / gram of the strain.

10. The composition of claim 8, wherein, The composition further comprises a fungicide, Preferably, the fungicide includes one or more of triazole fungicides, benzimidazole fungicides, imidazole fungicides, methoxy acrylate fungicides, cyano acrylate fungicides, amide fungicides, organic sulfur fungicides, organic chlorine fungicides, More preferably, the fungicide comprises one or more of tebuconazole, carbendazim, carpropamid, fluoroimid, iprodione, isopropothioamide, fluopyram, fenfuram, carboxin, oxycarboxin, thifluzamide, pyraclostrobin, bixafen, isopropothioamide, boscalid, cyprodinil, metconazole, simeconazole, silthiofam, penthiopyrad, fluazinam, pyraclostrobin, zoxamide, fluoxastrobin, prothioconazole and anilazodine, More preferably, the weight ratio of the Bacillus velezensis to the fungicide is (1-10):

1.

11. An inoculant characterized in that, The Bacillus velezensis strain according to any one of claims 1 to 3.

12. The bacterial agent of claim 11, wherein The fungicide is in the form of granules, wettable powder or water agent, Preferably, the fungicide is in the form of wettable powder, More preferably, the fungicide comprises, in parts by weight, 5-20% of the Bacillus velezensis strain, 0.5-2% of a dispersing agent, 0.5-2% of a wetting agent, and the balance of a filler.

13. A method for preparing the bacterial agent of claim 11 or 12, characterized by, The method comprises the following steps: obtaining a fermentation broth containing spores of the Bacillus velezensis strain; carrying out acidification treatment; filtering to obtain a filtrate, adding a stabilizer to the filtrate, and then carrying out spray drying to obtain strain powder; mixing the strain powder with a dispersing agent, a wetting agent and a filler to obtain the Bacillus velezensis strain fungicide.

14. The method of claim 13, wherein, The stabilizer is selected from trehalose, dextran, sucrose; and / or The acidification treatment adjusts the pH of the fermentation broth to 3.0-5.0; The dispersing agent is at least one of sodium lignosulfonate, calcium lignosulfonate, naphthalene sulfonate formaldehyde condensate and carboxylate salt polymer; The wetting agent is at least one of sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl laurate and alkyl naphthalene sulfonate.

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